Patent Application
20110009286
The present invention describes a molecular in vitro diagnosis of breast cancers. More particularly, the invention relates to the use of molecular probes for the diagnosis evaluation of breast tumors.
Breast Cancer is the most frequent cancer in women in western countries (40 000 new cases in France/year). Although several advances have been achieved in the last 10 years the mortality rates remain high (11 000 death/year in France). There is therefore a need for a new generation of advances in this disease. Two axes have allowed improvement of breast cancer survival rates: i. an earlier and more accurate diagnosis, ii. the development of adjuvant therapies.
Breast Cancer diagnosis is currently performed in a vast majority of cases by a preoperative core needle biopsy. This exam is currently considered as a level 1 recommendation in France (www.has-sante.fr). In selected centers that are experts in this field, a fine needle aspiration is used to perform breast cancer diagnosis. Overall, the preoperative diagnosis of breast cancer is currently allowed by microscopic assessment of samples obtained either fine needle aspiration (FNA) or core needle biopsy (CNB). Each of this approach presents some limitations that hamper the quality of care in patients who present a breast lesion. FNA is associated with an unacceptable rate of misdiagnosis. The rate of uncertain diagnosis is indeed around 30% (Oyama T, Koibuchi Y, McKee G. Core needle biopsy (CNB) as a diagnostic method for breast lesions: comparison with fine needle aspiration cytology (FNA). Breast Cancer. 2004; 11(4):339-42; Orell S R, Farshid G. False-positive reports in fine needle biopsy of breast lesions. Pathology. 2001 November; 33(4):428-36; Meunier M, Clough K. Fine needle aspiration cytology versus percutaneous biopsy of nonpalpable breast lesions. Eur J Radiol. 2002 April; 42(1):10-6). CNB exhibits better performance for diagnosis but is associated with some major limitations. First, since CNB is a complex procedure, the appointments are usually given several weeks after detection of breast lesions. This induces a delayed diagnosis and therefore treatment. With the increase of mass screening, this delay will probably increase over time. Second, the CNB is associated with clinical side effects. The rate of hematoma related to the procedure indeed range between 0.6 and 4% (Barreau B, Tastet S, Lakdja F, Henriques C, Valentin F, Labat M J, Dilhuydy M H. [Patients' information in percutaneous core breast biopsy] Bull Cancer. 2005 March; 92(3):257-65). In addition, CNB is associated is a more painful procedure as compared to FNA. Finally, CNB is an expensive procedure for a frequent disease (200 Euros/procedure). Overall, from these considerations rises the need for a diagnostic test performed on FNA samples that would be accurate, quick and cost-saving. This test could either substitute cytological exam of FNA sample or be performed in complement to this one. This test would allow an earlier, safer and cost-saving diagnosis procedure.
Regarding mass screening, mammogram is currently considered as the gold standard for mass screening. Nevertheless, this approach is limited by a high rate of false negative results, especially in young women and in aggressive tumors. In addition, mammogram is limited by the fact that a significant proportion of women do not perform the test. These considerations point out the need for a screening test that would be better or complementary to mammogram and that would be based on an easier procedure.
So, there is a need to provide test for the patients to diagnose and give a diagnosis of the status of the breast tumor detected.
Many methods based on the knowledge of the molecular mechanism of the breast tumor evolution have been extensively described. These methods describe in particular large-scale process for the determination of variation of expression of genes, or proteins, which can define a hallmark of the breast tumor evolution.
WO 2007/048978 describes methods and compositions for the detection of molecular markers in order to diagnose cancer. In particular, this document describes methods and compositions that can be used to detect the presence or evolution of a breast tumor in a patient.
However, this document discloses in particular micro-arrays for the evaluation of miss-regulated expressed genes, without interest for the expression of variants of genes.
WO 2004/065545 discloses a diagnostic and prognostic method of the breast cancer tumor status, wherein said method consists to compare the expression profile of at least 5 genes of a determined list of 70 genes to the expression profile of said at least 5 genes of a determined list of 70 genes of a control tumor.
However, this document never teaches that the disclosed method can detect splicing variant of the mentioned at least 5 genes chosen among the list of 70 genes to determine the breast cancer status of a tumor.
The setting-up of new arrays to analyse the large-scale expression of exons allows a highly accurate identification of differential molecular events between two conditions. This approach not only analyzes genes differentially expressed (as it is the case with conventional DNA array) but also the differential splicing events. Applied to breast cancer, this large scale analysis could identify differential expression of gene sequences between cancer and benign lesions. Based on these data, it would be possible to set-up molecular assays that could allow a molecular diagnosis of breast cancer based on FNA samples, that would allow a quick, accurate and cost-saving diagnosis. In addition, this approach could allow to set-up a molecular test for breast cancer screening based on blood samples, ductal lavage or breast sampling by FNA.
One aim of the invention is to provide a molecular diagnosis to determine the status of a breast tumor.
Another aim of the invention provides micro-arrays to determine the benign or malignant status of a breast tumor.
Another aim of the invention provides a method using the micro-array to determine the benign or malignant status of a breast tumor.
The invention also provides a kit for the breast cancer diagnosis.
The invention discloses the use of a multiplicity of polynucleotide probe sets, said multiplicity of polynucleotide probe sets consisting in a combination of pools of polynucleotide probe sets, each polynucleotide probe set containing at least one polynucleotide probe chosen among a library of nucleic acid sequences, each nucleic acid sequence specifically hybridizing with a gene, and/or at least one of its variants when present, said gene being chosen among the group of genes consisting in SEQ ID NO 5419 to SEQ ID NO 5693,
The invention discloses the use of a multiplicity of polynucleotide probe sets,
wherein
In the case of KRT17, LOC440421, KRT6C and KRT6A genes, said genes being respectively represented by SEQ ID NO 5431; 5689; 5651 and 5682, the above mentioned definition of the nucleic acid sequence of each polynucleotide probe of a given pool of polynucleotide probe set applies with the exception that:
The invention is based on the unexpected observation made by the inventors that the variation of expression of some genes, and their variants when they exist, may characterize the evolution of a diagnosed breast tumor. More particularly, the variants of the invention are the products of a differential splicing, or alternative splicing.
The inventors have demonstrated that the variation of expression of at least 12 determined genes of a group of 275 determined genes, and of variants of said at least 12 determined genes when they exist, hallmarks the benign or malignant status of a breast tumor.
Then, the invention describes the use of polynucleotide probes to characterize a benign versus malignant hallmark of a breast tumor, each polynucleotide probe hybridizing specifically with one gene, and at least one of its splicing variants when they exist, said gene splicing variant being able to be recognized by at least one polynucleotide probe.
In genetics, splicing is a modification of genetic information after transcription, in which introns of precursor messenger RNA (pre-mRNA) are removed and exons of precursor messenger RNA are joined. The splicing prepares the pre-mRNA to produce the mature messenger RNA (mRNA), which then undergoes translation as part of the protein synthesis to produce proteins.
Alternative splicing, in the sense of the invention, is the RNA splicing variation mechanism, in which the exons of the pre-mRNA are separated and reconnected so as to produce alternative ribonucleotide arrangements. In this way, alternative splicing uses genetic expression to facilitate the synthesis of a greater variety of transcripts.
When the pre-mRNA has been transcribed from the DNA, it includes several introns and exons. The exons which are retained in the mRNA are determined during the splicing process. The use of alternative splicing factors leads to a modification of the definition of a “gene” and imposes the variant notion.
For example a pre-mRNA containing 3 exons, if it is considered that variants must contain at least one exon of the gene and at least one exon missing of said gene, can hypothetically produce 6 different variants, as the result of alternative splicing.
In the invention, a gene is defined by the fact that it comprises the deoxyribonucleic acid sequence corresponding to the mRNA which results of the transcription of the DNA sequence, and after the splicing process. Then, the gene is characterized in that it comprises all the exons present in the genomic sequence of said gene.
All the 275 genes considered in the invention present a nucleic acid sequence chosen among the group consisting in SEQ ID NO 5419 to SEQ ID NO 5693.
In the invention, there are two alternatives for a gene:
In the case of a gene having no variant, this gene can is characterized in the invention by the presence of a target region. This target region corresponds to a nucleic acid sequence contained in the total nucleic acid sequence of the said gene.
In the case of a gene having one variant, this gene is defined by a target region, and its variant is defined by the same target region. The difference between the gene and its variant is preferably determined by the fact that the nucleic acid sequence of said gene is longer than the nucleic acid sequence of its variant.
This recognizing by one target region allows a redundancy of the representativity of said gene. Indeed, the target region contained in a gene having one variant is thus recognized at least twice, i.e. when the gene is present and when the variant of this gene is present. This redundancy enhances the specificity of detection of the gene expression variation.
In the case of a gene having at least two variants, this gene is defined by at least two regions, these two regions characterizing the gene. These regions are therefore present in gene. These regions, also called target regions correspond to nucleic acid sequences contained in the total nucleic acid sequence of said gene. Moreover, these target regions of said gene are located at a different position in the sequence of said gene.
The targets regions that are involved in the invention present a nucleic acid sequence chosen among the group consisting in SEQ ID NO 2939 to SEQ ID NO 4578.
According to the invention, a “variant” is defined as a polynucleotide molecule that differs from the reference polynucleotide molecule (the gene), but retains essential properties. The gene and its variants share similar polynucleotide sequences with, for example, 70% of nucleic acids identity, preferably 80% of nucleic acids identity, preferably 90% of nucleic acids identity and more preferably 95% of nucleic acids identity. The variants of the invention can be also considered as isoforms.
To be considered as a variant of a given gene, a polynucleotide sequence must contain at least one nucleotide sequence present in the said gene, this sequence corresponding to the target sequence defined above.
If a variant is the unique variant of a gene, this variant has the same target region as its gene, preferably with a nucleic acid sequence with a size that is shorter than the size of the nucleic acid sequence of said gene. Since said variant corresponds to a splicing product of a gene, it generally has one or more exons lacking that are present in said gene. Nevertheless, it is possible, in some rare examples, that a variant of a gene has a nucleic acid sequence with a size which is higher than the size of the nucleic acid sequence of said gene.
If a variant is a variant of a gene that has at least two variants, said variant has at least one difference in the presence of a nucleic acid sequence with respect to the given gene nucleic acid sequence. The specific combination of the presence of at least one target sequence and the absence of at least one target sequence defines the specific characteristic of a given variant. Therefore, each variant is defined by a “bar code” corresponding to the diversity of the presence and absence of all the target sequences present in a gene.
For example, if a gene contains 3 exons, all the nucleic acid sequences consisting in least one exon of said gene, and missing at least one of the other 2 exons of the said given gene, are considered as variants of said given gene.
In the invention, a variant of a gene is characterized in that it comprises at least on target region. These target regions corresponds to polynucleotidic sequences chosen among the group consisting in SEQ ID NO 2939 to SEQ ID NO 4578.
So, a given variant of a gene can be characterized with respect to the target sequence by the fact it contains at least a target region, present in the gene corresponding to said variant, and must is devoid of at least one target region, present in the gene corresponding to said variant, only in the case of a gene that has at least two variants.
As defined above, a variant of a gene having at least two variants is then characterized by a bar code which corresponds to a combination of the presence of at least one target region of the gene, corresponding to the variant, and the absence of at least one target region of the gene corresponding to the variant.
A variant can also be alternatively defined. This other definition is supported by the feature of the invention: the alternative splicing.
When a gene, constituted by at least 3 exons, is differentially spliced, in the corresponding variants, some exons are fused to exons which are not naturally fused in the sequence of the gene. For example, in a gene constituted by 3 exons E1, E2 and E3, one variant can be constituted by the fusion of E1 and E3. In the gene, the natural configuration corresponds to the sequence E1-E2-E3. Then the configuration observed in the variant, E1-E3, is considered as a non natural configuration.
In this case, variant presents a sequence that does not exist in the natural configuration of the gene. It generally corresponds to a junction of to distant exons, separated by at least one other exon. If the target region characterizing the variant corresponds to the junction between two exons, naturally distant in the normal configuration in the gene, said target region characterizes specifically said variant. This example is illustrated by
Example of genes having, or not, at least one variant are represented in
According to the invention, polynucleotide probes are used to detect the variation of expression of a gene, and/or its variants when they exist, to determine the status of a breast tumor.
Artificially, polynucleotide probes are grouped to form a polynucleotide probe set. The term “polynucleotide probe set” used in the invention defines a group of polynucleotide probes as described above. A given set of polynucleotide probe contains from one polynucleotide probe to several polynucleotide probes. Preferably, the polynucleotide probe set of the invention contains one polynucleotide probe to three or four polynucleotide probes. Thereby, polynucleotide probes regrouped in a polynucleotide probe set of the invention are able to hybridize to the same target region.
The polynucleotides probes are also brought together to form a “pool of polynucleotide probe sets”. The polynucleotides probes contained in the polynucleotide probe sets of a pool are able to specifically hybridize with nucleic acid sequences of a gene, and/or variants of said gene when they exist.
Alternatively, the polynucleotide probes contained in the polynucleotide probe sets of a pool are able to specifically hybridize neither with nucleic acid sequences of a different gene, nor with nucleic acid sequences of variants of said different gene, when they exist.
However, in few cases, the above definition should be modulated. In the case of genes KRT17 (SEQ ID NO 5431) and LOC440421 (SEQ ID NO 5689), these genes are two different genes, but share a high similarity/identity in their nucleic acid sequence. Thus, these two genes present some gene regions in their nucleic acid sequence which can be identical. If a target region is present in said gene region, therefore, the polynucleotide probe that recognize the target region contained in said gene region will be able to recognize the two genes.
A similar example applies with genes KRT6A (SEQ ID NO 5682) and KRT6C (SEQ ID NO 5651).
Then, polynucleotide probes of a pool of polynucleotide probe sets are able to characterize a gene and its variants. Moreover, a given gene, and its variants when they exist, can be detected only by the polynucleotide probes of the same pool of polynucleotide probe sets.
According to the invention, each gene, and its variants when present, can be detected by all the polynucleotide probes of a probe set. Moreover, each gene can be recognized by all the polynucleotide probes of at least one polynucleotide probe set, i.e. each gene and its variants can be defined by at least one polynucleotide probe set.
A variant can be detected by two polynucleotide probes belonging to two different polynucleotide probe sets, provided that they belong to the same pool of polynucleotide probe set.
The multiplicity, pool, polynucleotide probe set, and polynucleotide probe interconnection are represented in
Terms “library comprising or consisting of at least one copy each of the nucleic acids consisting of SEQ ID NO 1 to SEQ ID NO 2938” means that the library can contain one, or two, or three, or four, or five or six or seven or eight, or nine or ten copies of each polynucleotide probes represented by the nucleic acids consisting of SEQ ID NO 1 to SEQ ID NO 2938. Thus, the minimal library contains 2938 polynucleotides probes, and the maximal library contains 29380 polynucleotides probes. Preferably the preferred library of the invention contains 4582 polynucleotides probes.
In the invention, a “benign breast tumor” is any non-cancerous breast abnormality. Some (not all) benign conditions can signal an increased risk for breast cancer. The most common benign breast conditions include fibrocystic breast condition, benign breast tumors, and breast inflammation. Depending on the type of benign breast condition and the patient's medical situation, treatment may or may not be necessary.
In the invention, a “malignant breast tumor refers” to a malignant tumor that has developed from cells in the breast. Malignant tumors penetrate and destroy healthy body tissues. A group of cells within a tumor may also break away and spread to other parts of the body. Cells that spread from one region of the body into another are called metastases.
The term “base” is used to define the components of the DNA or RNA, i.e. deoxyribonucleotides and ribonucleotides respectively.
Terms “nucleic acids”, “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotidic sequence”, and “polynucleotide” are uniformly used to define a chain of bases that characterizing a DNA or an RNA molecule.
In the invention, “polynucleotide probe” represent a fragment of nucleic acid molecule, corresponding to DNA or RNA, with a variable length. The length of the polynucleotide probe is commonly comprised between 15 bases to 1000 bases, and preferably between 20 bases to 500 bases. Advantageously, the polynucleotide probes defined in the invention comprise from 20 bases to 30 bases. These polynucleotide probes are defined by their nucleic acid sequence.
In the invention, terms “multiplicity” and “combination” are used to define a group of polynucleotide probes sets and polynucleotide probes, respectively. Theses groups correspond to an ensemble of probes, or sets of probes, characterized in that they contains similar information.
According to the invention, each nucleic acid sequence corresponding to nucleic acid probe “specifically hybridizes with” a target region of the said gene. These terms mean that each polynucleotide probe is able to hybridize with a gene, and/or its variants when they exist, defined above. The polynucleotide probe thereby hybridizes to single-stranded nucleic acid molecule (gene and/or its variants, which are DNA or RNA) whose nucleic acid sequence allows base pairing due to complementarity between the nucleic acid sequence of the probe and the nucleic acid sequence of the gene and/or variants of said gene. The base pairing (A-T;G-C) is a concept well known by the skilled man in the art.
Each polynucleotide probe is able to hybridize with one gene and/or at least one variant of said gene, in a specific region of said variant called target region.
Said target region is then defined as the region allowing the base pairing between polynucleotide probe and a gene and/or its variants, when they exist.
Thereby, each polynucleotide probe is able to define a gene and/or at least one variant of said gene, when they exist.
According to the invention, each polynucleotide probe specifically hybridizes with a specific nucleic acid sequence contained in the target region. Moreover, each polynucleotide probes can recognizes a sequence overlapping partially with the sequence recognized by another probe of the same polynucleotide probe set.
To sum up, in the invention each gene, or reference transcript, can be expressed differentially in the form of variants. These variants differ from each other by the presence or absence or the variation of a sequence of a specific region: the target region. This region is liable to be recognized by one to three polynucleotide probes, said polynucleotide probes hybridizing with the same target region being brought together in a polynucleotide probe set.
Then, the use of the multiplicity of polynucleotide probe sets of the invention allows to detect the variation of expression of at least 12 determined genes consisting in SEQ ID NO 5421, SEQ ID NO 5423, SEQ ID NO 5432, SEQ ID NO 5434, SEQ ID NO 5486, SEQ ID NO 5491, SEQ ID NO 5525, SEQ ID NO 5552, SEQ ID NO 5599, SEQ ID NO 5652, SEQ ID NO 5654 and SEQ ID NO 5683, and their variants when they exist, of the group of determined genes constituted by genes of the group constituted by SEQ ID NO 5419 to SEQ ID NO 5693, and a skill man in the art can determine if the breast tumor is malignant or benign.
Thereby, if the expression of a given gene and/or at least one of its variants belonging to the group of 12 determined genes corresponds to a “profile” similar to that of a benign tumor, the tumor of the patient will be considered as evolving to a benign status. In contrast, the expression of a gene and/or at least one of its variant belonging to the group of 12 genes corresponds to that of a “profile” similar to a malignant tumor, it will be considered that the tumor is a malignant tumor.
In one embodiment, the invention discloses the use of a multiplicity of polynucleotide probe sets, wherein the multiplicity of polynucleotide probe sets consisting in a combination of pools of polynucleotide probe sets chosen among 1640 probe sets of polynucleotides, each polynucleotide probe set containing at least one polynucleotide probe chosen among a library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938, each nucleic acid sequence hybridizing with a gene, and/or at least one of its variants when present, said gene being chosen among the group of genes consisting in SEQ ID NO 5419 to SEQ ID NO 5693,
for determining the variation of expression a gene, and its variants when present, of said sub-group in order to diagnose the benign or malignant state of a breast tumor.
In one preferred embodiment, the invention discloses the use of a multiplicity of polynucleotide probe sets defined above, for determining the variation of the expression of at least 20 determined genes of a group of genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693,
The invention resides in the unexpected observation that the variation of expression of a group of at least 20 determined genes chosen among 275 determined genes is sufficient to determine the malignant or the benign status of a breast tumor.
In another preferred embodiment the invention discloses the use of a multiplicity of polynucleotide probe sets as above defined, for determining the variation of the expression of at least 35 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
The invention also resides in the unexpected observation that the variation of expression of a group of at least 35 determined genes chosen among 275 determined genes is sufficient to determine the malignant or the benign status of a breast tumor.
In another preferred embodiment the invention discloses the use of a multiplicity of polynucleotide probe sets previously defined, for determining the variation of the expression of at least 43 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693,
The invention also resides in the unexpected observation that the variation of expression of a group of at least 43 determined genes chosen among 275 determined genes is sufficient to determine the malignant or the benign status of a breast tumor.
In another preferred embodiment the invention discloses the use of a multiplicity of polynucleotide probe sets such as defined above, for determining the variation of the expression of at least 206 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
The invention also resides in the unexpected observation that the variation of expression of a group of at least 206 determined genes chosen among 275 determined genes is sufficient to determine the malignant or the benign status of a breast tumor, and provide the best results.
In another preferred embodiment the invention discloses the use of a multiplicity of polynucleotide probe sets such as above defined, for determining the variation of the expression of 275 determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693.
The invention also resides in the unexpected observation that the variation of expression of 275 determined genes is sufficient to determine the malignant or the benign status of a breast tumor.
Then, in an advantageous embodiment, the invention describes the use of a multiplicity of polynucleotide probe sets that allow the detection of variation of expression 12 genes, and their variants when they exist, said variation defining the benign or malignant hallmark of the breast tumor.
More particularly, the 12 genes, and their variants when they exist, are represented by their nucleic acid sequences consisting in SEQ ID NO 5421, SEQ ID NO 5423, SEQ ID NO 5432, SEQ ID NO 5434, SEQ ID NO 5486, SEQ ID NO 5491, SEQ ID NO 5525, SEQ ID NO 5552, SEQ ID NO 5599, SEQ ID NO 5652, SEQ ID NO 5654 and SEQ ID NO 5683 and are specifically recognized by their target sequences.
In one preferred embodiment, the invention relates to the use of a multiplicity of polynucleotide probe sets for determining the variation the gene expression as defined above, and of at least one variant of said genes when present, said variant being represented by the nucleic acid sequences SEQ ID NO 4579 to SEQ ID NO 5418 and SEQ ID NO 5694, the correspondence between each variant and its corresponding gene being represented in Table 1A-E and in Table 6.
Thus, according to the invention the inventors have demonstrated that the determination of the variation of expression of at least 12 genes chosen among a group of 275 genes, and of at least one variant of each at least 12 genes give a good information about the benign or malignant status of a breast tumor, without important false positives.
Also, others aspects of the invention demonstrate that the study of the variation of expression of:
In one particular embodiment, the variation of expression of all the 275 determined genes and at least one of their variant allows the diagnostic.
In another particular embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of at least 12 determined genes of the group of 275 determined genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of the variants of said at least 12 determined genes, wherein the correspondence between genes and their variants is indicated in the following table 1A.
Table 1A represents the correspondences between the group of 12 determined genes and their corresponding variants.
In another particular embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of at least 20 determined genes of the group of 275 genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of the variants of said at least 20 determined genes, wherein the correspondence between genes and their variants is indicated in the following table 1B.
Table 1B represents the correspondences between the group of 20 determined genes and their corresponding variants.
In another preferred embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of at least 35 determined genes of the group of 275 determined genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of the variants of said at least 35 determined genes, wherein the correspondence between genes and their variants is indicated in the following table 1C.
Table 1C represents the correspondences between the group of 35 determined genes and their corresponding variants.
In other preferred embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of at least 43 determined genes of the group of 275 determined genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of the variants of said at least 43 determined genes, wherein the correspondence between genes and their variant s is indicated in the following table 1D.
Table 1D represents the correspondences between the group of 43 determined genes and their corresponding variants.
In another preferred embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of at least 206 determined genes of the group of 275 determined genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of the variants of said at least 206 determined genes, wherein the correspondence between genes and their variants is indicated in the following table 1E.
Table 1E represents the correspondences between the group of 206 determined genes and their corresponding variants.
In another particular embodiment, the invention relates to the use as defined above, for the determination of the variation of expression of 275 determined genes represented by SEQ ID NO 5419 to SEQ ID NO 5693, and of at least one of their variants when they exist, wherein the correspondence between genes and their variants is indicated in the following table 6.
Table 6 summarizes the correspondence between all the sequences disclosed in the invention. In this table:
In another preferred embodiment, the invention relates to the use of a multiplicity of polynucleotide probe sets for determining the variation of the gene expression as defined above, and of its variants when present, wherein target regions are characterized by the polynucleotide sequences SEQ ID NO 2939 to 4578, each target region of each gene, and of each variant when they exist, being represented in Table 6.
For example, in the invention, for detecting the variation of expression of the group of at least 12 determined genes of the group of 275 genes, each of the 12 genes has the following target region:
With Table 6, knowing gene and variants, it is easy to identify the sequence of the corresponding target region.
In another particular embodiments, the invention discloses the use of a multiplicity of polynucleotide probe sets for determining the variation of the gene expression as defined above, and/or of their variants when present, wherein each polynucleotide probe contained in a polynucleotide probe set is represented by the nucleic acid sequence of the library consisting in SEQ ID NO 1 to SEQ ID NO 2938, the correspondence between each polynucleotide probe and its corresponding target region, said target region corresponding to a gene and its variants when present, being represented in Table 6.
For example, in the invention, for detecting the variation of expression of the group of at least 12 determined genes of the group of 275 genes, each of the 12 genes is recognized by the following polynucleotide probes:
Using Table 6, and knowing genes and variants, it is easy to identify the sequence of the corresponding target region and probes.
The invention also relates to the use of a multiplicity of at least 50 polynucleotide probe sets chosen among 1640 polynucleotide probe sets, the nucleic acid sequences of the polynucleotide probes contained in said 50 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 136; 137; 138; 157; 158; 159; 167; 168; 169; 171; 172; 173; 177; 312; 313; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 333; 334; 335; 336; 339; 340; 341; 345; 346; 347; 348; 353; 360; 361; 364; 365; 366; 367; 368; 369; 370; 374; 375; 790; 791; 792; 793; 794; 795; 969; 972; 981; 1081; 1082; 1083; 1129; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1185; 1186; 1194; 1198; 1199; 1200; 1203; 1262; 1263; 1264; 1267; 1268; 1269; 1519; 1520; 1521; 1522; 1523; 1524; 1534; 1535; 1536; 1544; 1545; 1546; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 2026; 2027; 2028; 2179; 2180 and 2181,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
for the determination of the variation of expression of:
In one preferred embodiment, the invention relates to the use as defined above, wherein the polynucleotide probes contained in said at least 50 polynucleotide probe sets are as mentioned above and wherein SEQ ID NOs 167; 168; 177; 312; 313; 327; 328; 329; 331; 364; 365; 366; 368; 370; 1185; 1186; 1194; 1523; 1524; 1551; 1552 and 1553 are present twice, SEQ ID NO 316 is present in three copies and SEQ ID NO 315 is present in 5 copies.
Thus, in all the 50 polynucleotide probe sets mentioned above, 143 polynucleotide probes are present, the above mentioned polynucleotide probes being present at least once, and in particular SEQ ID NOs 167; 168; 177; 312; 313; 327; 328; 329; 331; 364; 365; 366; 368; 370; 1185; 1186; 1194; 1523; 1524; 1551; 1552 and 1553 are present twice, SEQ ID NO 316 is present in three copies and SEQ ID NO 315 is present in 5 copies.
The above mentioned polynucleotide probes contained in the 50 nucleotides probe sets specifically recognized the above mentioned gene and their variant when present via target region, the correspondence being deduced from Table 6.
In one preferred embodiment, the invention relates to the use of a multiplicity of at least 100 polynucleotide probe sets as defined above, the nucleic acid sequence of the polynucleotides probes contained in said 100 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NO 120; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 312; 313; 314; 315; 316; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 363; 364; 365; 366; 367; 368; 369; 370; 371; 374; 375; 376; 378; 379; 380; 381; 555; 556; 557; 558; 560; 561; 562; 566; 567; 788; 789; 790; 791; 792; 793; 794; 795; 800; 801; 802; 821; 822; 823; 969; 971; 972; 981; 1056; 1057; 1058; 1075; 1076; 1077; 1081; 1082; 1083; 1087; 1088; 1089; 1103; 1104; 1105; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1130; 1131; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1198; 1199; 1200; 1201; 1202; 1203; 1207; 1262; 1263; 1264; 1267; 1268; 1269; 1509; 1510; 1511; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1544; 1545; 1546; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1561; 1562; 1682; 1683; 1684; 1776; 1777; 1778; 2023; 2024; 2025; 2026; 2027; 2028; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2762; 2763; 2764; 2860; 2861; 2862; 2883; 2884 and 2885,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
for the determination of the variation of expression of
In one preferred embodiment, the invention relates to the use as defined above, wherein the polynucleotide probes contained in said at least 100 polynucleotide probe sets are as mentioned above and wherein SEQ ID NOs 167; 168; 177; 312; 313; 316; 317; 327; 329; 330; 353; 361; 364; 365; 366; 367; 368; 369; 370; 821; 822; 823; 1129; 1164; 1185; 1186; 1187; 1188; 1189; 1203; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 328; 331; 1162; 1194; 1519; 1520; 1521 and 1524 are present in three copies and SEQ ID NO 315 is present in 6 copies.
Thus, in all the 100 polynucleotide probe sets mentioned above, 282 polynucleotide probes are present, the above mentioned polynucleotide probes being present at least once, and in particular SEQ ID NOs 167; 168; 177; 312; 313; 316; 317; 327; 329; 330; 353; 361; 364; 365; 366; 367; 368; 369; 370; 821; 822; 823; 1129; 1164; 1185; 1186; 1187; 1188; 1189; 1203; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 328; 331; 1162; 1194; 1519; 1520; 1521 and 1524 are present in three copies and SEQ ID NO 315 is present in 6 copies.
The above mentioned polynucleotide probes contained in the 100 nucleotides probe sets specifically recognized the above mentioned gene and their variant when present via target region, the correspondence being deduced from Table 6.
In another preferred embodiment, the invention relates to the use of a multiplicity of at least 150 polynucleotide probe sets as previously defined, the nucleic acid sequence of the polynucleotides probes contained in said 150 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 4; 5; 6; 120; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 197; 248; 254; 255; 305; 306; 307; 312; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 362; 363; 364; 365; 366; 367; 367; 368; 369; 370; 371; 374; 375; 376; 377; 378; 379; 380; 381; 513; 514; 515; 552; 553; 554; 555; 556; 557; 558; 559; 560; 561; 562; 566; 567; 783; 784; 785; 788; 789; 790; 791; 792; 793; 794; 795; 796; 797; 798; 799; 800; 801; 802; 803; 804; 805; 806; 807; 808; 812; 813; 814; 818; 821; 822; 823; 968; 969; 970; 971; 972; 981; 1056; 1057; 1058; 1061; 1062; 1063; 1064; 1065; 1066; 1067; 1068; 1069; 1070; 1071; 1075; 1076; 1077; 1080; 1081; 1082; 1083; 1087; 1088; 1089; 1103; 1104; 1105; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1130; 1131; 1139; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1174; 1175; 1176; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1197; 1198; 1199; 1200; 1201; 1202; 1203; 1204; 1205; 1206; 1207; 1262; 1263; 1264; 1267; 1268; 1269; 1284; 1285; 1315; 1469; 1470; 1471; 1509; 1510; 1511; 1515; 1516; 1517; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1537; 1538; 1539; 1544; 1545; 1546; 1550; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1558; 1559; 1560; 1561; 1562; 1580; 1581; 1582; 1671; 1682; 1683; 1684; 1685; 1686; 1690; 1691; 1692; 1776; 1777; 1778; 1788; 1789; 1790; 1890; 1891; 1892; 1952; 1953; 1954; 1963; 2023; 2024; 2025; 2026; 2027; 2028; 2065; 2067; 2088; 2089; 2090; 2091; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2196; 2201; 2229; 2230; 2231; 2536; 2537; 2538; 2576; 2577; 2578; 2599; 2600; 2762; 2763; 2764; 2774; 2775; 2776; 2860; 2861; 2862; 2883; 2884 and 2885,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
for the determination of the variation of expression of
In one preferred embodiment, the invention relates to the use as defined above, wherein the polynucleotide probes contained in said at least 150 polynucleotide probe sets are as mentioned above and wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 969; 1056; 1057; 1058; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1469; 1470; 1471; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1194; 1519; 1520; 1521; 1524 are present in three copies, SEQ ID NO 1162 is present in 4 copies and SEQ ID NO 315 is present in 6 copies.
Thus, in all the 150 polynucleotide probe sets mentioned above, 422 polynucleotide probes are present, the above mentioned polynucleotide probes being present at least once, and in particular SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 969; 1056; 1057; 1058; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1469; 1470; 1471; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1194; 1519; 1520; 1521; 1524 are present in three copies, SEQ ID NO 1162 is present in 4 copies and SEQ ID NO 315 is present in 6 copies.
The above mentioned polynucleotide probes contained in the 150 nucleotides probe sets specifically recognized the above mentioned gene and their variant when present via target region, the correspondence being deduced from Table 6.
A preferred embodiment of the invention relates to the use of a multiplicity of at least 200 polynucleotide probe sets as defined above, the nucleic acid sequence of the polynucleotide probes contained in said 200 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 4; 5; 6; 120; 121; 122; 123; 130; 131; 132; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 195; 196; 197; 198; 199; 200; 248; 254; 255; 305; 306; 307; 312; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 362; 363; 364; 365; 366; 367; 368; 369; 370; 371; 374; 375; 376; 377; 378; 379; 380; 381; 513; 514; 515; 552; 553; 554; 555; 556; 557; 558; 559; 560; 561; 562; 563; 564; 565; 566; 567; 783; 784; 785; 788; 789; 790; 791; 792; 793; 794; 795; 796; 797; 798; 799; 800; 801; 802; 803; 804; 805; 806; 807; 808; 809; 810; 811; 812; 813; 814; 815; 816; 817; 818; 821; 822; 823; 824; 825; 826; 840; 841; 845; 846; 847; 868; 968; 969; 970; 971; 972; 973; 974; 975; 976; 977; 981; 1056; 1057; 1058; 1059; 1060; 1061; 1062; 1063; 1064; 1065; 1066; 1067; 1068; 1069; 1070; 1071; 1075; 1076; 1077; 1080; 1081; 1082; 1083; 1084; 1085; 1086; 1087; 1088; 1089; 1094; 1095; 1096; 1103; 1104; 1105; 1110; 1111; 1112; 1114; 1115; 1116; 1123; 1124; 1125; 1126; 1127; 1128; 1129; 1130; 1131; 1139; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1174; 1175; 1176; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1197; 1198; 1199; 1200; 1201; 1202; 1203; 1204; 1205; 1206; 1207; 1229; 1230; 1242; 1243; 1244; 1245; 1250; 1251; 1252; 1262; 1263; 1264; 1265; 1266; 1267; 1268; 1269; 1270; 1271; 1272; 1284; 1285; 1315; 1335; 1407; 1408; 1469; 1470; 1471; 1509; 1510; 1511; 1512; 1513; 1514; 1515; 1516; 1517; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1537; 1538; 1539; 1544; 1545; 1546; 1550; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1558; 1559; 1560; 1561; 1562; 1563; 1564; 1580; 1581; 1582; 1586; 1587; 1588; 1671; 1682; 1683; 1684; 1685; 1686; 1687; 1688; 1689; 1690; 1691; 1692; 1743; 1744; 1745; 1776; 1777; 1778; 1788; 1789; 1790; 1890; 1891; 1892; 1896; 1897; 1898; 1952; 1953; 1954; 1963; 1974; 2023; 2024; 2025; 2026; 2027; 2028; 2061; 2062; 2063; 2065; 2067; 2068; 2072; 2083; 2084; 2085; 2088; 2089; 2090; 2091; 2092; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2196; 2201; 2229; 2230; 2231; 2381; 2382; 2383; 2536; 2537; 2538; 2576; 2577; 2578; 2599; 2600; 2601; 2602; 2603; 2762; 2763; 2764; 2774; 2775; 2776; 2778; 2779; 2783; 2794; 2860; 2861; 2862; 2883; 2884 and 2885,
for the determination of the variation of expression of
In one preferred embodiment, the invention relates to the use as defined above, wherein the polynucleotide probes contained in said at least 200 polynucleotide probe sets are as mentioned above and wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 367; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 845; 968; 970; 972; 1056; 1057; 1058; 1085; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1523; 1551; 1552; 1553; 1557; 1586; 1587; 1588; 1952; 1953; 1954; 2088; 2090; 2091; 2762; 2763 and 2764 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1284; 1285; 1469; 1470; 1471; 1519; 1520; 1521; 1524; 2065; 2067 and 2089 are present in three copies, SEQ ID NO 1194 is present in 4 copies and SEQ ID NOs 315 and 1162 are present in 6 copies.
Thus, in all the 200 polynucleotide probe sets mentioned above, 565 polynucleotide probes are present, the above mentioned polynucleotide probes being present at least once, and in particular SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 367; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 845; 968; 970; 972; 1056; 1057; 1058; 1085; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1523; 1551; 1552; 1553; 1557; 1586; 1587; 1588; 1952; 1953; 1954; 2088; 2090; 2091; 2762; 2763 and 2764 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1284; 1285; 1469; 1470; 1471; 1519; 1520; 1521; 1524; 2065; 2067 and 2089 are present in three copies, SEQ ID NO 1194 is present in 4 copies and SEQ ID NOs 315 and 1162 are present in 6 copies.
The above mentioned polynucleotide probes contained in the 200 nucleotides probe sets specifically recognized the above mentioned gene and their variant when present via target region, the correspondence being deduced from Table 6.
The multiplicities mentioned above are considered as the “Top Polynucleotide Probe Sets”.
In another preferred embodiment, the invention relates to the use of a multiplicity of at least 1228 polynucleotide probe sets as defined above, containing 3985 polynucleotide probes, the nucleic acid sequence of the polynucleotide probes contained in said 1228 polynucleotide probe sets being represented in the first 1228 lines of the Table 7,
for the determination of the variation of expression of for determining the variation of the expression of
SEQ ID Nos 5420; 5421; 5422; 5423; 5425; 5426; 5427; 5428; 5429; 5430; 5431; 5432; 5434; 5435; 5436; 5437; 5438; 5439; 5440; 5441; 5442; 5443; 5444; 5445; 5446; 5447; 5449; 5450; 5451; 5452; 5453; 5454; 5455; 5456; 5457; 5459; 5460; 5461; 5462; 5463; 5465; 5467; 5468; 5470; 5473; 5475; 5476; 5479; 5480; 5482; 5484; 5485; 5486; 5487; 5488; 5489; 5490; 5491; 5492; 5493; 5495; 5496; 5497; 5498; 5499; 5502; 5503; 5505; 5506; 5507; 5508; 5509; 5511; 5512; 5513; 5515; 5516; 5517; 5518; 5519; 5520; 5523; 5524; 5525; 5526; 5528; 5529; 5530; 5531; 5532; 5533; 5534; 5536; 5538; 5539; 5540; 5543; 5544; 5545; 5546; 5547; 5549; 5550; 5552; 5553; 5554; 5556; 5558; 5559; 5560; 5562; 5563; 5564; 5565; 5566; 5567; 5568; 5569; 5570; 5571; 5573; 5574; 5575; 5576; 5577; 5578; 5581; 5582; 5585; 5587; 5588; 5590; 5591; 5593; 5594; 5595; 5597; 5598; 5599; 5600; 5601; 5602; 5603; 5604; 5605; 5606; 5608; 5609; 5610; 5611; 5612; 5613; 5615; 5616; 5617; 5618; 5622; 5623; 5624; 5625; 5627; 5629; 5630; 5631; 5632; 5633; 5634; 5635; 5637; 5638; 5639; 5640; 5641; 5642; 5643; 5644; 5645; 5647; 5648; 5649; 5651; 5652; 5653; 5654; 5656; 5657; 5658; 5660; 5661; 5665; 5667; 5669; 5672; 5673; 5675; 5676; 5677; 5678; 5680; 5681; 5683; 5684; 5686; 5687; 5689 and 5692, and the corresponding variants being represented in table 1E,
in order to diagnose in vitro or ex vivo the benign or malignant state of a breast tumor.
Thus, in all the 1228 polynucleotide probe sets mentioned above, 3427 polynucleotide probes are present.
Also, a preferred embodiment of the invention discloses the use of a multiplicity of 1640 polynucleotide probe sets as defined above, the nucleic acid sequence of the polynucleotide probes contained in said 1640 polynucleotide probe sets being represented in Table 7,
for the determination of the variation of expression of
Thus, in all the 1640 polynucleotide probe sets mentioned above, 4586 polynucleotide probes are present.
In another embodiment, the invention discloses the use of a multiplicity of polynucleotide probe sets, wherein the sub-group of genes is constituted by 12 to 259 genes chosen among the group of genes consisting in SEQ ID NO 5419 to SEQ ID NO 5677.
The sub-groups of genes contain at least 12 genes, as described above. The sub-group of genes can also contain advantageously 19 genes, or advantageously 32 genes, or advantageously 40 genes, or advantageously 214 genes or more advantageously 259 genes.
According to the invention, each gene of the sub-groups of genes, and/or its variants when they exist, are specifically recognized by at least one polynucleotide probe belonging to a polynucleotide probe set of a pool of polynucleotide probe sets.
In a more particular embodiment, the invention relates to the use of a multiplicity of polynucleotide probe sets, wherein the sub-group of genes is constituted by the 259 genes of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5693.
The inventors have unexpectedly demonstrated that a group of 259 genes, and/or their variants when they exist, give some powerful information to precisely determine the benign or malignant status of a breast tumor.
In the group of 259 genes, each gene, and its variants when they exist is specifically recognized by at least one polynucleotide probe belonging to a polynucleotide probe set of a pool of polynucleotide probe sets.
In another advantageous embodiment, the invention discloses the use of a multiplicity of polynucleotide probe sets, wherein the polynucleotide probe sets are chosen among at least 50 polynucleotide probe sets, particularly at least 100 polynucleotide probe sets, more particularly at least 150 polynucleotide probe sets, more particularly at least 200 polynucleotide probe sets, more particularly at least 1228 polynucleotide probe sets, and more particularly 1640 polynucleotide probe sets of the library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938.
The multiplicity of polynucleotide probe sets is of at least 50 polynucleotide probe sets, and particularly at least 100 polynucleotide probe sets, more particularly at least 150 polynucleotide probe sets, and more advantageously at least 200 polynucleotide probe sets.
These multiplicities are considered as the “Top Polynucleotide Probe Sets”.
The Top 50 polynucleotide probe sets contains a combination of polynucleotide probes characterized by SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 177, SEQ ID NO 312, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 317, SEQ ID NO 324, SEQ ID NO 326, SEQ ID NO 327, SEQ ID NO 329, SEQ ID NO 330, SEQ ID NO 331SEQ ID NO 333, SEQ ID NO 335, SEQ ID NO 336, SEQ ID NO 339, SEQ ID NO 341, SEQ ID NO 345, SEQ ID NO 347, SEQ ID NO 353, SEQ ID NO 360, SEQ ID NO 361, SEQ ID NO 364, SEQ ID NO 366, SEQ ID NO 367, SEQ ID NO 369, SEQ ID NO 790, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 795, SEQ ID NO 969, SEQ ID NO 972, SEQ ID NO 981, SEQ ID NO 1081, SEQ ID NO 1083, SEQ ID NO 1129, SEQ ID NO 1140, SEQ ID NO 1141, SEQ ID NO 1151, SEQ ID NO 1158, SEQ ID NO 1159, SEQ ID NO 1162, SEQ ID NO 1165, SEQ ID NO 1166, SEQ ID NO 1167, SEQ ID NO 1169, SEQ ID NO 1170, SEQ ID NO 1171, SEQ ID NO 1185, SEQ ID NO 1186, SEQ ID NO 1194, SEQ ID NO 1198, SEQ ID NO 1200, SEQ ID NO 1203, SEQ ID NO 1262, SEQ ID NO 1264, SEQ ID NO 1267, SEQ ID NO 1269, SEQ ID NO 1520, SEQ ID NO 1522, SEQ ID NO 1523, SEQ ID NO 1524, SEQ ID NO 1534, SEQ ID NO 1536, SEQ ID NO 1544, SEQ ID NO 1546, SEQ ID NO 1551, SEQ ID NO 1553, SEQ ID NO 1554, SEQ ID NO 1556, SEQ ID NO 1557, SEQ ID NO 2026, SEQ ID NO 2028, SEQ ID NO 2179 and SEQ ID NO 2181.
The Top 100 polynucleotide probe set contains a combination of polynucleotide probes characterized by SEQ ID NO 120, SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 312, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 317, SEQ ID NO 324, SEQ ID NO 326, SEQ ID NO 327, SEQ ID NO 328, SEQ ID NO 329, SEQ ID NO 330, SEQ ID NO 331, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 333, SEQ ID NO 335, SEQ ID NO 336, SEQ ID NO 337, SEQ ID NO 338, SEQ ID NO 339, SEQ ID NO 341, SEQ ID NO 345, SEQ ID NO 347, SEQ ID NO 349, SEQ ID NO 350, SEQ ID NO 351, SEQ ID NO 352, SEQ ID NO 353, SEQ ID NO 354, SEQ ID NO 355, SEQ ID NO 356, SEQ ID NO 360, SEQ ID NO 361, SEQ ID NO 363, SEQ ID NO 364, SEQ ID NO 364, SEQ ID NO 366, SEQ ID NO 367, SEQ ID NO 369, SEQ ID NO 369, SEQ ID NO 371, SEQ ID NO 376, SEQ ID NO 378, SEQ ID NO 379, SEQ ID NO 380, SEQ ID NO 381, SEQ ID NO 555, SEQ ID NO 556, SEQ ID NO 557, SEQ ID NO 558, SEQ ID NO 560, SEQ ID NO 561, SEQ ID NO 562, SEQ ID NO 566, SEQ ID NO 567, SEQ ID NO 788, SEQ ID NO 789, SEQ ID NO 790, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 795, SEQ ID NO 800, SEQ ID NO 801, SEQ ID NO 802, SEQ ID NO 821, SEQ ID NO 822, SEQ ID NO 823, SEQ ID NO 969, SEQ ID NO 971, SEQ ID NO 972, SEQ ID NO 981, SEQ ID NO 1056, SEQ ID NO 1057, SEQ ID NO 1058, SEQ ID NO 1075, SEQ ID NO 1076, SEQ ID NO 1077, SEQ ID NO 1081, SEQ ID NO 1083, SEQ ID NO 1087, SEQ ID NO 1088, SEQ ID NO 1089, SEQ ID NO 1103, SEQ ID NO 1104, SEQ ID NO 1105, SEQ ID NO 1114, SEQ ID NO 1115, SEQ ID NO 1116, SEQ ID NO 1123, SEQ ID NO 1124, SEQ ID NO 1125, SEQ ID NO 1129, SEQ ID NO 1130, SEQ ID NO 1131, SEQ ID NO 1140, SEQ ID NO 1141, SEQ ID NO 1142, SEQ ID NO 1143, SEQ ID NO 1151, SEQ ID NO 1158, SEQ ID NO 1159, SEQ ID NO 1162, SEQ ID NO 1163, SEQ ID NO 1163, SEQ ID NO 1164, SEQ ID NO 1165, SEQ ID NO 1166, SEQ ID NO 1167, SEQ ID NO 1169, SEQ ID NO 1170, SEQ ID NO 1171, SEQ ID NO 1172, SEQ ID NO 1172, SEQ ID NO 1180, SEQ ID NO 1181, SEQ ID NO 1185, SEQ ID NO 1186, SEQ ID NO 1187, SEQ ID NO 1188, SEQ ID NO 1189, SEQ ID NO 1189, SEQ ID NO 1190, SEQ ID NO 1191, SEQ ID NO 1192, SEQ ID NO 1193, SEQ ID NO 1194, SEQ ID NO 1195, SEQ ID NO 1196, SEQ ID NO 1198, SEQ ID NO 1200, SEQ ID NO 1201, SEQ ID NO 1202, SEQ ID NO 1203, SEQ ID NO 1207, SEQ ID NO 1262, SEQ ID NO 1264, SEQ ID NO 1267, SEQ ID NO 1269, SEQ ID NO 1509, SEQ ID NO 1510, SEQ ID NO 1511, SEQ ID NO 1519, SEQ ID NO 1520, SEQ ID NO 1520, SEQ ID NO 1521, SEQ ID NO 1522, SEQ ID NO 1523, SEQ ID NO 1524, SEQ ID NO 1524, SEQ ID NO 1528, SEQ ID NO 1529, SEQ ID NO 1534, SEQ ID NO 1536, SEQ ID NO 1544, SEQ ID NO 1546, SEQ ID NO 1551, SEQ ID NO 1553, SEQ ID NO 1554, SEQ ID NO 1556, SEQ ID NO 1557, SEQ ID NO 1561, SEQ ID NO 1562, SEQ ID NO 1682, SEQ ID NO 1683, SEQ ID NO 1684, SEQ ID NO 1776, SEQ ID NO 1777, SEQ ID NO 1778, SEQ ID NO 2023, SEQ ID NO 2024, SEQ ID NO 2025, SEQ ID NO 2026, SEQ ID NO 2028, SEQ ID NO 2173, SEQ ID NO 2174, SEQ ID NO 2175, SEQ ID NO 2176, SEQ ID NO 2177, SEQ ID NO 2178, SEQ ID NO 2179, SEQ ID NO 2181, SEQ ID NO 2762, SEQ ID NO 2763, SEQ ID NO 2764, SEQ ID NO 2860, SEQ ID NO 2861, SEQ ID NO 2862, SEQ ID NO 2883, SEQ ID NO 2884 and SEQ ID NO 2885.
The Top 150 polynucleotide probe set contains a combination of polynucleotide probes characterized by polynucleotide probes chosen among SEQ ID NO 1 to SEQ ID NO 2938.
The Top 200 polynucleotide probe set contains a combination of polynucleotide probes characterized by polynucleotide probes chosen among SEQ ID NO 1 to SEQ ID NO 2938.
According to the invention, the multiplicities used can also be represented by a number of at least 300 polynucleotide probe sets, more particularly at least 400 polynucleotide probe sets, more particularly at least 500 polynucleotide probe sets, more particularly at least 600 polynucleotide probe sets, more particularly at least 700 polynucleotide probe sets, more particularly at least 800 polynucleotide probe sets, more particularly at least 900 polynucleotide probe sets, more particularly at least 1000 polynucleotide probe sets, more particularly at least 1100 polynucleotide probe sets, more particularly at least 1228 polynucleotide probe sets, more particularly at least 1400 polynucleotide probe sets, and more particularly 1640 polynucleotide probe sets.
All the above-mentioned multiplicities of polynucleotide probe sets contain polynucleotides probes chosen among the library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938.
According to the invention, the 1228 polynucleotide probe sets are an advantageous multiplicity of probe sets defined to diagnose the benign versus malignant status of a breast tumor.
In another embodiment, the invention also relates to the use of a multiplicity of polynucleotide probe sets, wherein polynucleotide probe sets are chosen among at least 50 polynucleotide probe sets, particularly at least 100 polynucleotide probe sets, more particularly at least 150 polynucleotide probe sets, more particularly at least 200 polynucleotide probe sets, more particularly at least 1228 polynucleotide probe sets, and more particularly 1640 polynucleotide probe sets of the library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938,
and wherein the sub-group of genes is constituted by the 259 genes of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677.
According to the invention, the benign versus malignant status of a breast tumor can be determined by the use of 2938 polynucleotide probes.
In an advantageous embodiment, the invention describes the use of a multiplicity of polynucleotide probe sets, wherein a variant of a given gene is chosen among the group of variants consisting in SEQ ID NO 4579 to SEQ ID NO 5418.
According to the invention, each gene can have:
In an advantageous embodiment, the invention discloses the use of a multiplicity of polynucleotide probe sets, each polynucleotide probe set containing at least one polynucleotide probe, each probe being able to specifically hybridize with at least one gene, and/or its variants when they exist, to determine the benign or malignant status of a breast tumor.
More particularly, the invention discloses the use of 2938 polynucleotides probes, assembled in polynucleotide probe sets, said polynucleotides probes being able to detect the variation of expression of at least one gene chosen among 259 genes, and/or its variants when present. According to the invention, the variation of expression of at least 839 variants and the variation of expression 259 genes can be detected by the use of the multiplicities of polynucleotide probe sets described above.
The invention also discloses a library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938.
This library consists in all the polynucleotide probes that can be regrouped in 1640 representative probe sets of the invention. Then, the library consists in a group that contains 2938 polynucleotide probes.
The invention advantageously discloses a multiplicity of polynucleotide probes sets chosen among the nucleic acid sequences of the library described above, wherein the multiplicity of polynucleotide probe sets represents a combination of pool of polynucleotide probe sets able to specifically hybridize with 214 genes, and/or their variants when they exist, chosen among SEQ ID NO 5419 to SEQ ID NO 5677.
The group of polynucleotide probes represents a combination of 1228 polynucleotide probe sets. The group allows to detect the variation of expression of 214 genes and/or their variants when they exist.
The invention advantageously discloses a multiplicity of polynucleotide probe sets chosen among the nucleic acid sequences of the library according to claim 8, wherein the multiplicity of polynucleotide probe sets represents a combination of pool of polynucleotide probe sets able to specifically hybridize with 40 genes, and/or their variants when they exist, chosen among SEQ ID NO 5419 to SEQ ID NO 5677.
The group of polynucleotide probes represents a combination of 200 polynucleotide probe sets. The group allows to detect the variation of expression of 40 genes and/or their variants when they exist.
The invention more advantageously discloses a multiplicity of polynucleotide probe sets chosen among the nucleic acid sequences of the library according to claim 8, wherein the multiplicity of polynucleotide probe sets represents a combination of pool of polynucleotide probe sets able to specifically hybridize with 32 genes, and/or their variants when they exist, chosen among SEQ ID NO 5419 to SEQ ID NO 5677.
The group of polynucleotide probes represents a combination of 150 polynucleotide probe sets. The group allows to detect the variation of expression of 32 genes and/or their variants when they exist.
The invention more advantageously discloses multiplicity of polynucleotide probe sets chosen among the nucleic acid sequences of the library according to claim 8, wherein the multiplicity of polynucleotide probe sets represents a combination of pool of polynucleotide probe sets able to specifically hybridize with 19 genes, and/or their variants when they exist, chosen among SEQ ID NO 5419 to SEQ ID NO 5677.
According to the invention, the set of polynucleotide probe being able to specifically hybridize with 19 genes, and/or their variants when they exist, comprises the polynucleotide probes consisting in SEQ ID NO 120, SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 312, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 317, SEQ ID NO 324, SEQ ID NO 326, SEQ ID NO 327, SEQ ID NO 328, SEQ ID NO 329, SEQ ID NO 330, SEQ ID NO 331, SEQ ID NO 331, SEQ ID NO 332, SEQ ID NO 333, SEQ ID NO 335, SEQ ID NO 336, SEQ ID NO 337, SEQ ID NO 338, SEQ ID NO 339, SEQ ID NO 341, SEQ ID NO 345, SEQ ID NO 347, SEQ ID NO 349, SEQ ID NO 350, SEQ ID NO 351, SEQ ID NO 352, SEQ ID NO 353, SEQ ID NO 354, SEQ ID NO 355, SEQ ID NO 356, SEQ ID NO 360, SEQ ID NO 361, SEQ ID NO 363, SEQ ID NO 364, SEQ ID NO 364, SEQ ID NO 366, SEQ ID NO 367, SEQ ID NO 369, SEQ ID NO 369, SEQ ID NO 371, SEQ ID NO 376, SEQ ID NO 378, SEQ ID NO 379, SEQ ID NO 380, SEQ ID NO 381, SEQ ID NO 555, SEQ ID NO 556, SEQ ID NO 557, SEQ ID NO 558, SEQ ID NO 560, SEQ ID NO 561, SEQ ID NO 562, SEQ ID NO 566, SEQ ID NO 567, SEQ ID NO 788, SEQ ID NO 789, SEQ ID NO 790, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 795, SEQ ID NO 800, SEQ ID NO 801, SEQ ID NO 802, SEQ ID NO 821, SEQ ID NO 822, SEQ ID NO 823, SEQ ID NO 969, SEQ ID NO 971, SEQ ID NO 972, SEQ ID NO 981, SEQ ID NO 1056, SEQ ID NO 1057, SEQ ID NO 1058, SEQ ID NO 1075, SEQ ID NO 1076, SEQ ID NO 1077, SEQ ID NO 1081, SEQ ID NO 1083, SEQ ID NO 1087, SEQ ID NO 1088, SEQ ID NO 1089, SEQ ID NO 1103, SEQ ID NO 1104, SEQ ID NO 1105, SEQ ID NO 1114, SEQ ID NO 1115, SEQ ID NO 1116, SEQ ID NO 1123, SEQ ID NO 1124, SEQ ID NO 1125, SEQ ID NO 1129, SEQ ID NO 1130, SEQ ID NO 1131, SEQ ID NO 1140, SEQ ID NO 1141, SEQ ID NO 1142, SEQ ID NO 1143, SEQ ID NO 1151, SEQ ID NO 1158, SEQ ID NO 1159, SEQ ID NO 1162, SEQ ID NO 1163, SEQ ID NO 1163, SEQ ID NO 1164, SEQ ID NO 1165, SEQ ID NO 1166, SEQ ID NO 1167, SEQ ID NO 1169, SEQ ID NO 1170, SEQ ID NO 1171, SEQ ID NO 1172, SEQ ID NO 1172, SEQ ID NO 1180, SEQ ID NO 1181, SEQ ID NO 1185, SEQ ID NO 1186, SEQ ID NO 1187, SEQ ID NO 1188, SEQ ID NO 1189, SEQ ID NO 1189, SEQ ID NO 1190, SEQ ID NO 1191, SEQ ID NO 1192, SEQ ID NO 1193, SEQ ID NO 1194, SEQ ID NO 1195, SEQ ID NO 1196, SEQ ID NO 1198, SEQ ID NO 1200, SEQ ID NO 1201, SEQ ID NO 1202, SEQ ID NO 1203, SEQ ID NO 1207, SEQ ID NO 1262, SEQ ID NO 1264, SEQ ID NO 1267, SEQ ID NO 1269, SEQ ID NO 1509, SEQ ID NO 1510, SEQ ID NO 1511, SEQ ID NO 1519, SEQ ID NO 1520, SEQ ID NO 1520, SEQ ID NO 1521, SEQ ID NO 1522, SEQ ID NO 1523, SEQ ID NO 1524, SEQ ID NO 1524, SEQ ID NO 1528, SEQ ID NO 1529, SEQ ID NO 1534, SEQ ID NO 1536, SEQ ID NO 1544, SEQ ID NO 1546, SEQ ID NO 1551, SEQ ID NO 1553, SEQ ID NO 1554, SEQ ID NO 1556, SEQ ID NO 1557, SEQ ID NO 1561, SEQ ID NO 1562, SEQ ID NO 1682, SEQ ID NO 1683, SEQ ID NO 1684, SEQ ID NO 1776, SEQ ID NO 1777, SEQ ID NO 1778, SEQ ID NO 2023, SEQ ID NO 2024, SEQ ID NO 2025, SEQ ID NO 2026, SEQ ID NO 2028, SEQ ID NO 2173, SEQ ID NO 2174, SEQ ID NO 2175, SEQ ID NO 2176, SEQ ID NO 2177, SEQ ID NO 2178, SEQ ID NO 2179, SEQ ID NO 2181, SEQ ID NO 2762, SEQ ID NO 2763, SEQ ID NO 2764, SEQ ID NO 2860, SEQ ID NO 2861, SEQ ID NO 2862, SEQ ID NO 2883, SEQ ID NO 2884 and SEQ ID NO 2885
The group of polynucleotide probes represents a combination of 100 polynucleotide probe sets. The group allows to detect the variation of expression of 19 genes and/or their variants when they exist.
The invention advantageously discloses a multiplicity of polynucleotide probe sets chosen among the nucleic acid sequences of the library according to claim 8, wherein the multiplicity of polynucleotide probe sets represents a combination of pool of polynucleotide probe sets able to specifically hybridize with 12 genes, and/or their variants when they exist, chosen among SEQ ID NO 5419 to SEQ ID NO 5677.
According to the invention, the set of polynucleotide probe being able to specifically hybridize with 12 genes, and/or their variants when they exist, comprises the polynucleotide probes consisting in SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 177, SEQ ID NO 312, SEQ ID NO 314, SEQ ID NO 315, SEQ ID NO 317, SEQ ID NO 324, SEQ ID NO 326, SEQ ID NO 327, SEQ ID NO 329, SEQ ID NO 330, SEQ ID NO 331SEQ ID NO 333, SEQ ID NO 335, SEQ ID NO 336, SEQ ID NO 339, SEQ ID NO 341, SEQ ID NO 345, SEQ ID NO 347, SEQ ID NO 353, SEQ ID NO 360, SEQ ID NO 361, SEQ ID NO 364, SEQ ID NO 366, SEQ ID NO 367, SEQ ID NO 369, SEQ ID NO 790, SEQ ID NO 792, SEQ ID NO 793, SEQ ID NO 795, SEQ ID NO 969, SEQ ID NO 972, SEQ ID NO 981, SEQ ID NO 1081, SEQ ID NO 1083, SEQ ID NO 1129, SEQ ID NO 1140, SEQ ID NO 1141, SEQ ID NO 1151, SEQ ID NO 1158, SEQ ID NO 1159, SEQ ID NO 1162, SEQ ID NO 1165, SEQ ID NO 1166, SEQ ID NO 1167, SEQ ID NO 1169, SEQ ID NO 1170, SEQ ID NO 1171, SEQ ID NO 1185, SEQ ID NO 1186, SEQ ID NO 1194, SEQ ID NO 1198, SEQ ID NO 1200, SEQ ID NO 1203, SEQ ID NO 1262, SEQ ID NO 1264, SEQ ID NO 1267, SEQ ID NO 1269, SEQ ID NO 1520, SEQ ID NO 1522, SEQ ID NO 1523, SEQ ID NO 1524, SEQ ID NO 1534, SEQ ID NO 1536, SEQ ID NO 1544, SEQ ID NO 1546, SEQ ID NO 1551, SEQ ID NO 1553, SEQ ID NO 1554, SEQ ID NO 1556, SEQ ID NO 1557, SEQ ID NO 2026, SEQ ID NO 2028, SEQ ID NO 2179 and SEQ ID NO 2181.
The group of polynucleotide probes represents a combination of 50 polynucleotide probe sets. The group allows to detect the variation of expression of 12 genes and/or their variants when they exist.
The invention also discloses a multiplicity of 50 polynucleotide probe sets chosen among 1640 polynucleotide probe sets as defined above.
Also, the invention relates to a multiplicity of at least 50 polynucleotide probe sets chosen among 1640 polynucleotide probe sets, the nucleic acid sequences of the polynucleotide probes contained in said 50 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 136; 137; 138; 157; 158; 159; 167; 168; 169; 171; 172; 173; 177; 312; 313; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 333; 334; 335; 336; 339; 340; 341; 345; 346; 347; 348; 353; 360; 361; 364; 365; 366; 367; 368; 369; 370; 374; 375; 790; 791; 792; 793; 794; 795; 969; 972; 981; 1081; 1082; 1083; 1129; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1185; 1186; 1194; 1198; 1199; 1200; 1203; 1262; 1263; 1264; 1267; 1268; 1269; 1519; 1520; 1521; 1522; 1523; 1524; 1534; 1535; 1536; 1544; 1545; 1546; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 2026; 2027; 2028; 2179; 2180 and 2181,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 327; 328; 329; 331; 364; 365; 366; 368; 370; 1185; 1186; 1194; 1523; 1524; 1551; 1552 and 1553 are present twice, SEQ ID NO 316 is present in three copies and SEQ ID NO 315 is present in 5 copies.
In one preferred embodiment, the invention also discloses a multiplicity of 100 polynucleotide probe sets chosen among 1640 polynucleotide probe sets as defined above.
Also, the invention relates to a multiplicity of at least 100 polynucleotide probe sets as above defined, the nucleic acid sequences of the polynucleotides probes contained in said 100 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NO 120; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 312; 313; 314; 315; 316; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 363; 364; 365; 366; 367; 368; 369; 370; 371; 374; 375; 376; 378; 379; 380; 381; 555; 556; 557; 558; 560; 561; 562; 566; 567; 788; 789; 790; 791; 792; 793; 794; 795; 800; 801; 802; 821; 822; 823; 969; 971; 972; 981; 1056; 1057; 1058; 1075; 1076; 1077; 1081; 1082; 1083; 1087; 1088; 1089; 1103; 1104; 1105; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1130; 1131; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1198; 1199; 1200; 1201; 1202; 1203; 1207; 1262; 1263; 1264; 1267; 1268; 1269; 1509; 1510; 1511; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1544; 1545; 1546; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1561; 1562; 1682; 1683; 1684; 1776; 1777; 1778; 2023; 2024; 2025; 2026; 2027; 2028; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2762; 2763; 2764; 2860; 2861; 2862; 2883; 2884 and 2885,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 316; 317; 327; 329; 330; 353; 361; 364; 365; 366; 367; 368; 369; 370; 821; 822; 823; 1129; 1164; 1185; 1186; 1187; 1188; 1189; 1203; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 328; 331; 1162; 1194; 1519; 1520; 1521 and 1524 are present in three copies and SEQ ID NO 315 is present in 6 copies.
In another preferred embodiment, the invention also discloses a multiplicity of 150 polynucleotide probe sets chosen among 1640 polynucleotide probe sets as defined above.
In other word, the invention relates to a multiplicity of at least 150 polynucleotide probe sets previously defined, the nucleic acid sequences of the polynucleotides probes contained in said 150 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 4; 5; 6; 120; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 197; 248; 254; 255; 305; 306; 307; 312; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 362; 363; 364; 365; 366; 367; 367; 368; 369; 370; 371; 374; 375; 376; 377; 378; 379; 380; 381; 513; 514; 515; 552; 553; 554; 555; 556; 557; 558; 559; 560; 561; 562; 566; 567; 783; 784; 785; 788; 789; 790; 791; 792; 793; 794; 795; 796; 797; 798; 799; 800; 801; 802; 803; 804; 805; 806; 807; 808; 812; 813; 814; 818; 821; 822; 823; 968; 969; 970; 971; 972; 981; 1056; 1057; 1058; 1061; 1062; 1063; 1064; 1065; 1066; 1067; 1068; 1069; 1070; 1071; 1075; 1076; 1077; 1080; 1081; 1082; 1083; 1087; 1088; 1089; 1103; 1104; 1105; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1130; 1131; 1139; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1174; 1175; 1176; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1197; 1198; 1199; 1200; 1201; 1202; 1203; 1204; 1205; 1206; 1207; 1262; 1263; 1264; 1267; 1268; 1269; 1284; 1285; 1315; 1469; 1470; 1471; 1509; 1510; 1511; 1515; 1516; 1517; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1537; 1538; 1539; 1544; 1545; 1546; 1550; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1558; 1559; 1560; 1561; 1562; 1580; 1581; 1582; 1671; 1682; 1683; 1684; 1685; 1686; 1690; 1691; 1692; 1776; 1777; 1778; 1788; 1789; 1790; 1890; 1891; 1892; 1952; 1953; 1954; 1963; 2023; 2024; 2025; 2026; 2027; 2028; 2065; 2067; 2088; 2089; 2090; 2091; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2196; 2201; 2229; 2230; 2231; 2536; 2537; 2538; 2576; 2577; 2578; 2599; 2600; 2762; 2763; 2764; 2774; 2775; 2776; 2860; 2861; 2862; 2883; 2884 and 2885,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 969; 1056; 1057; 1058; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1469; 1470; 1471; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1194; 1519; 1520; 1521; 1524 are present in three copies, SEQ ID NO 1162 is present in 4 copies and SEQ ID NO 315 is present in 6 copies.
Another preferred embodiment of the invention relates to a multiplicity of 200 polynucleotide probe sets chosen among 1640 polynucleotide probe sets as defined above.
Indeed, a preferred embodiment of the invention discloses a multiplicity of at least 200 polynucleotide probe sets defined above, the nucleic acid sequences of the polynucleotide probes contained in said 200 polynucleotide probe sets being the following nucleic acid sequences: SEQ ID NOs 4; 5; 6; 120; 121; 122; 123; 130; 131; 132; 136; 137; 138; 154; 155; 156; 157; 158; 159; 167; 168; 169; 171; 172; 173; 174; 175; 176; 177; 195; 196; 197; 198; 199; 200; 248; 254; 255; 305; 306; 307; 312; 313; 314; 315; 316; 317; 324; 325; 326; 327; 328; 329; 330; 331; 332; 333; 334; 335; 336; 337; 338; 339; 340; 341; 345; 346; 347; 348; 349; 350; 351; 352; 353; 354; 355; 356; 360; 361; 362; 363; 364; 365; 366; 367; 368; 369; 370; 371; 374; 375; 376; 377; 378; 379; 380; 381; 513; 514; 515; 552; 553; 554; 555; 556; 557; 558; 559; 560; 561; 562; 563; 564; 565; 566; 567; 783; 784; 785; 788; 789; 790; 791; 792; 793; 794; 795; 796; 797; 798; 799; 800; 801; 802; 803; 804; 805; 806; 807; 808; 809; 810; 811; 812; 813; 814; 815; 816; 817; 818; 821; 822; 823; 824; 825; 826; 840; 841; 845; 846; 847; 868; 968; 969; 970; 971; 972; 973; 974; 975; 976; 977; 981; 1056; 1057; 1058; 1059; 1060; 1061; 1062; 1063; 1064; 1065; 1066; 1067; 1068; 1069; 1070; 1071; 1075; 1076; 1077; 1080; 1081; 1082; 1083; 1084; 1085; 1086; 1087; 1088; 1089; 1094; 1095; 1096; 1103; 1104; 1105; 1110; 1111; 1112; 1114; 1115; 1116; 1123; 1124; 1125; 1126; 1127; 1128; 1129; 1130; 1131; 1139; 1140; 1141; 1158; 1159; 1160; 1161; 1162; 1163; 1164; 1165; 1166; 1167; 1168; 1169; 1170; 1171; 1172; 1174; 1175; 1176; 1180; 1181; 1185; 1186; 1187; 1188; 1189; 1190; 1191; 1192; 1193; 1194; 1195; 1196; 1197; 1198; 1199; 1200; 1201; 1202; 1203; 1204; 1205; 1206; 1207; 1229; 1230; 1242; 1243; 1244; 1245; 1250; 1251; 1252; 1262; 1263; 1264; 1265; 1266; 1267; 1268; 1269; 1270; 1271; 1272; 1284; 1285; 1315; 1335; 1407; 1408; 1469; 1470; 1471; 1509; 1510; 1511; 1512; 1513; 1514; 1515; 1516; 1517; 1519; 1520; 1521; 1522; 1523; 1524; 1528; 1529; 1534; 1535; 1536; 1537; 1538; 1539; 1544; 1545; 1546; 1550; 1551; 1552; 1553; 1554; 1555; 1556; 1557; 1558; 1559; 1560; 1561; 1562; 1563; 1564; 1580; 1581; 1582; 1586; 1587; 1588; 1671; 1682; 1683; 1684; 1685; 1686; 1687; 1688; 1689; 1690; 1691; 1692; 1743; 1744; 1745; 1776; 1777; 1778; 1788; 1789; 1790; 1890; 1891; 1892; 1896; 1897; 1898; 1952; 1953; 1954; 1963; 1974; 2023; 2024; 2025; 2026; 2027; 2028; 2061; 2062; 2063; 2065; 2067; 2068; 2072; 2083; 2084; 2085; 2088; 2089; 2090; 2091; 2092; 2173; 2174; 2175; 2176; 2177; 2178; 2179; 2180; 2181; 2196; 2201; 2229; 2230; 2231; 2381; 2382; 2383; 2536; 2537; 2538; 2576; 2577; 2578; 2599; 2600; 2601; 2602; 2603; 2762; 2763; 2764; 2774; 2775; 2776; 2778; 2779; 2783; 2794; 2860; 2861; 2862; 2883; 2884 and 2885,
each polynucleotide probe set containing from 1 to 4 of said polynucleotide probes,
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 367; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 845; 968; 970; 972; 1056; 1057; 1058; 1085; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1523; 1551; 1552; 1553; 1557; 1586; 1587; 1588; 1952; 1953; 1954; 2088; 2090; 2091; 2762; 2763 and 2764 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1284; 1285; 1469; 1470; 1471; 1519; 1520; 1521; 1524; 2065; 2067 and 2089 are present in three copies, SEQ ID NO 1194 is present in 4 copies and SEQ ID NOs 315 and 1162 are present in 6 copies.
Another particular embodiment of the invention relates to a multiplicity of 1228 polynucleotide probe sets chosen among 1640 polynucleotide probe sets as defined above.
The invention, in a particular embodiment, relates to a multiplicity of at least 1228 polynucleotide probe sets as defined above, containing 3985 polynucleotide probes, the nucleic acid sequences of the polynucleotide probes contained in said 1228 polynucleotide probe sets being represented in the first 1228 lines of the Table 7.
The invention discloses, in another preferred embodiment, a multiplicity 1640 polynucleotide probe sets as defined above.
The invention related to a multiplicity of 1640 polynucleotide probe sets as previously defined, the nucleic acid sequences of the polynucleotide probes contained in said 1640 polynucleotide probe sets being represented in Table 7.
The invention also discloses a micro-array comprising or consisting in a multiplicity of polynucleotide probe sets as defined above.
Thus, the invention discloses a micro-array comprising the library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938, or a multiplicity of polynucleotide probe sets:
The microarray refers to nucleic acid molecule covalently attached to an inert chemical surface, such as coated glass slides or gene chips, and to which a mobile DNA target is hybridized. All the commonly used surfaces can also serve as a support.
The invention discloses a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject, by determining the variation of the expression of a gene, and/or at least one of its variants when present, of a sub-group comprising at least 12 genes among the genes of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677, comprising:
Also, the invention relates to a method for the determination, preferably in vitro and/or ex vivo, of the benign or malignant state of a breast tumor in a biological sample from a subject, by determining the variation of the expression of
comprising:
wherein
The invention also describes a method that allows the determination of the status, benign or malignant, of a breast tumor.
In the method of the invention, a biological sample corresponding to a breast tumor is used as a source of nucleic acid molecule. Said biological sample refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be one of any biological tissue. Frequently the sample could be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, FNA sample, CNB sample, ductal lavage and blood sample. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
The subject from which biological sample is provided is commonly a woman, afflicted, or suspected to be afflicted by a breast tumor. The subject can also be a man, with, or suspected to have, a breast cancer. By extension, all the mammals can be diagnosed by the method provides by the invention, with the proviso that they could develop a breast tumor.
The extraction of the nucleic acid molecules of the samples is managed by a routine protocol used in the art. Advantageously, nucleic acid molecules extracted from the biological sample are RNA.
Then, the method of the invention consists in contacting nucleic acid molecules extracted from the biological sample of a subject, with a multiplicity of polynucleotide probe sets. Each polynucleotide probe set contains at least one polynucleotide probe.
The contact between the polynucleotide probe set, and more particularly the polynucleotide probes contained in each polynucleotide probe set, allows to form a nucleic acid complex between the nucleic acid molecule corresponding to a gene, and/or its variants when they exist, and a polynucleotide probe.
Preferably, before contacting the polynucleotide probes with the nucleic acid molecules, nucleic acid molecules are labeled with any know labeling agents (radioisotopes, enzymes, fluorescent molecule . . . ). The hybridization is made according a standard procedure, by modulating if necessary saline concentration and temperature. The protocol used for hybridization is well known by the skilled man in the art.
The presence and amount of the formed nucleic acid complex is detected, by the detection of hybridized nucleic acid molecules which have been labeled with labeling agent, with a specific detection method fitting to the used labeled agent.
According to the invention, each probe set that have hybridized with labeled nucleic acid molecules allows to attribute a value of hybridization. The given value is dependant of the labeling agent.
The compilation of all the values of hybridization allows to establish a profile that corresponds to the specific feature of the tumor assayed. This result is considered as a hallmark of the tumor assayed.
The tumor profile is compared to a reference profile, in particular profile of tumors deriving from biological sample of non afflicted by breast cancer subjects, or derived from biological sample of a subject afflicted by a benign or a malignant breast tumor. Alternatively, the tumor profile assayed can be compared with tumor profile of patients afflicted by benign breast tumor or malignant breast tumor. Typically, the comparison is made by using software commonly used in this type of comparison, and well known and described in the art.
From the comparison between tumor profile and reference profile, it is possible to determine if the breast tumor derived from the biological sample of a patient is a benign tumor or a malignant tumor.
In an advantageous embodiment, the invention discloses a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject, wherein the determination of the variation of the expression of a gene, and/or at least one of its variants when present, of a sub-group comprising at least 12 genes among the genes of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677, comprises:
In one preferred embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject as defined above, by determining the variation of the expression of at least 20 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
said 20 determined genes being represented by the following nucleotidic sequences: SEQ ID Nos 5421; 5423; 5432; 5434; 5457; 5486; 5491; 5513; 5516; 5525; 5532; 5552; 5599; 5616; 5624; 5643; 5652; 5654; 5673 and 5683.
In one other preferred embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject such as defined above, by 35 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
SEQ ID Nos 5421; 5423; 5431; 5432; 5434; 5440; 5457; 5462; 5470; 5486; 5491; 5505; 5513; 5516; 5525; 5532; 5546; 5552; 5594; 5599; 5601; 5603; 5616; 5618; 5624; 5627; 5630; 5643; 5645; 5652; 5654; 5660; 5669; 5673 and 5683.
Another preferred embodiment of the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject as previously defined, by determining the variation of the expression of at least 43 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
Another particular embodiment of the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject as defined above, by determining the variation of the expression of at least 206 determined genes of a group of determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693
In another preferred embodiment, the invention discloses a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject such as defined above, by determining the variation of the expression of 275 determined genes represented by the nucleotidic sequences SEQ ID NO: 5419 to SEQ ID NO: 5693.
Another particular embodiment of the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject such as defined above, wherein said variant being represented by the nucleic acid sequences SEQ ID NO 4579 to SEQ ID NO 5418 and SEQ ID NO 5694, the correspondence between each variant and its corresponding gene being represented in Table 1A-E and Table 6.
In still another embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject as defined above, wherein target regions are characterized by the polynucleotide sequences SEQ ID NO 2939 to 4578, the each target region of each gene, and of each variant when they exist, being represented in Table 6.
In one other preferred embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject such as previously defined, wherein each polynucleotide probe contained in a polynucleotide probe set is represented by the nucleic acid sequence of the library consisting in SEQ ID NO 1 to SEQ ID NO 2938,
the correspondence between each polynucleotide probe and its corresponding target region, said target region corresponding to a gene and its variants when present, being represented in Table 6.
In another advantageous embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject, wherein the sub-group of genes is constituted by 12 to 259 genes chosen among the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677.
In another embodiment, the invention discloses a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor described above, wherein the sub-group of genes is constituted by the 259 genes of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677.
In a more particular embodiment, the invention relates to a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor in a biological sample from a subject, wherein said set of polynucleotide probes are chosen among the group consisting in:
at least 50 polynucleotide probe sets, particularly at least 100 polynucleotide probe sets, more particularly at least 150 polynucleotide probes sets, more particularly at least 200 polynucleotide probe sets, more particularly at least 1228 polynucleotide probe sets and more particularly 1640 polynucleotide probe sets of the library of nucleic acid sequences consisting in SEQ ID NO 1 to SEQ ID NO 2938.
The sub-groups of 50, 100, 150, 200 polynucleotide probe sets are defined as the Top sub-groups allowing to determine the benign or malignant status of the tumor.
It is also disclosed in the invention that the multiplicity of probe sets can be constituted by at least 300 polynucleotide probe sets, more particularly at least 400 polynucleotide probe sets, more particularly at least 500 polynucleotide probe sets, more particularly at least 600 polynucleotide probe sets, more particularly at least 700 polynucleotide probe sets, more particularly at least 800 polynucleotide probe sets, more particularly at least 900 polynucleotide probe sets, more particularly at least 1000 polynucleotide probe sets, more particularly at least 1100 polynucleotide probe sets, more particularly at least 1228 polynucleotide probe sets, more particularly at least 1400 polynucleotide probe sets, and more particularly 1640 polynucleotide probe sets.
All the polynucleotide probes contained in the polynucleotide probe sets used in the method of the invention are chosen among a library of nucleic acid sequences consisting SEQ ID NO 1 to SEQ ID NO 2938.
In one embodiment, the invention discloses a method described above, wherein a variant of a given gene is chosen among the group of variants consisting in SEQ ID NO 4579 to SEQ ID NO 5418.
The method of the invention allows to detect the variation of expression genes, and their variants when they exist, said genes being characterized in that they consist in the nucleic acid sequences chosen among the group consisting in SEQ ID NO 5419 to SEQ ID NO 5677 and said variants being characterized in that they consist in the nucleic acid sequence chosen among the group consisting in SEQ ID NO 4579 to SEQ ID NO 5418.
In the invention, it is also disclosed a method for the in vitro and/or ex vivo determination of the benign or malignant state of a breast tumor, wherein the variation of expression of a gene, and/or at least one of its variants when present, said gene being chosen in the group consisting in SEQ ID NO 5419 to SEQ ID NO 5693, is
Each gene expression, and expression of its variants if they exist, revealed by the method of the invention corresponds to an expression product of a gene of the group consisting in SEQ ID NO 5419 to SEQ ID NO 5693.
These variants are normally expressed in a subject non afflicted by breast cancer. These variants are also expressed in the biological of subject afflicted by benign breast cancer or malignant breast cancer. Each variant have a specific expression level corresponding to the state of the breast cancer. Each variant can then be overexpressed compare to the same variant expressed in a normal subject, or under expressed comparing to the same variant expressed in a normal subject.
The genes, and/or their variants are up-regulated, preferably when their expression levels are more than 1.7 fold higher than the expression levels of said genes in a patient non afflicted by a breast tumor, and significantly different (preferably with a significant coefficient value of less than 0.05). The genes, and/or their variants are down-regulated, preferably when their expression levels are less than 1.7 fold lower than the expression levels of said genes in a patient non afflicted by a breast tumor, and significantly different (preferably with a significant coefficient value of less than 0.05).
In a particular embodiment, the invention discloses a method described above, wherein the nucleic acid molecules from the biological sample of the subject are mRNA/or cDNA, said mRNA resulting from the expression of the genes consisting in SEQ ID NO 5419 to SEQ ID NO 5693, and/or at least one of their variants when present, represented by the nucleotidic sequences SEQ ID NO 4579 to SEQ ID NO 5418 and SEQ ID NO 5694.
In the invention, nucleic acid molecules are extracted from the biological sample of the subject, prior to be contacted with the set of polynucleotide probes, immobilized in a support.
The extraction and preparation of said nucleic acid molecules from the biological sample of the subject is made with a routine protocol commonly used by a skilled man in the art.
More particularly, the RNA are extracted from the biological sample, eventually, mRNA are isolated from the total RNA. In the case of the use of cDNA, mRNA are reverse-transcripted with a standard procedure.
The invention relates to a kit for the in vitro and/or ex vivo determination of benign or malignant status of a breast tumor, comprising:
The invention relates to a kit for the in vitro and/or ex vivo determination of benign or malignant status of a breast tumor, comprising the following group of polynucleotide probes:
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 327; 328; 329; 331; 364; 365; 366; 368; 370; 1185; 1186; 1194; 1523; 1524; 1551; 1552 and 1553 are present twice, SEQ ID NO 316 is present in three copies and SEQ ID NO 315 is present in 5 copies, or
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 316; 317; 327; 329; 330; 353; 361; 364; 365; 366; 367; 368; 369; 370; 821; 822; 823; 1129; 1164; 1185; 1186; 1187; 1188; 1189; 1203; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 328; 331; 1162; 1194; 1519; 1520; 1521 and 1524 are present in three copies and SEQ ID NO 315 is present in 6 copies, or
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 969; 1056; 1057; 1058; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1469; 1470; 1471; 1523; 1551; 1552 and 1553 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1194; 1519; 1520; 1521; 1524 are present in three copies, SEQ ID NO 1162 is present in 4 copies and SEQ ID NO 315 is present in 6 copies, or
preferably, wherein SEQ ID NOs 167; 168; 177; 312; 313; 317; 327; 329; 330; 353; 361; 363; 364; 365; 366; 367; 369; 370; 371; 558; 567; 796; 797; 801; 806; 807; 808; 813; 814; 821; 822; 823; 845; 968; 970; 972; 1056; 1057; 1058; 1085; 1114; 1115; 1116; 1123; 1124; 1125; 1129; 1141; 1163; 1164; 1185; 1186; 1187; 1188; 1189; 1199; 1203; 1523; 1551; 1552; 1553; 1557; 1586; 1587; 1588; 1952; 1953; 1954; 2088; 2090; 2091; 2762; 2763 and 2764 are present twice, SEQ ID NOs 316; 328; 331; 368; 788; 1284; 1285; 1469; 1470; 1471; 1519; 1520; 1521; 1524; 2065; 2067 and 2089 are present in three copies, SEQ ID NO 1194 is present in 4 copies and SEQ ID NOs 315 and 1162 are present in 6 copies, or
The invention also relates to
Preferably in the kit of the invention, the micro-array comprises 1228 polynucleotide probe sets that represent the best multiplicity of polynucleotide probe sets to characterize efficiently the benign or malignant status of the breast tumor of a subject.
Optionally, the kit of the invention may contain protocol and molecules to label nucleic acid molecules used to be contacted with the micro-array. Also, the kit of the invention may contain protocol and material for the extraction and purification of the nucleic acid molecules from the biological sample of a subject.
The contain of the kit is not limited to the described materials and protocols described above, and may contain any other material or procedures that can help a skilled man in the art to use the kit of the invention.
The following examples illustrate the invention, but the invention is not limited to these examples.
56652 probes (8%) presented a different intensity between malignant and benign conditions (adjusted p value<0.05). Of these, 37858 were exon-probes (B, T, and F) and 18794 were junction-probes (C, D, E). Of the 37858 exon-probes (B, T, F), 17441 were higher in malignant as opposed to benign diseases. The 17441 exon-probes (B, T, F) that were found higher in malignant condition referred to 12091 exons and 3391 genes. Of the 17441 exon-probes increased in malignant tumors, 1656 presented more than a two fold increased intensity in malignant conditions as compared to benign tumors. Heatmap of intensities for these 1656 probes is reported in
The level of gene expression was then determined as reported in patients and methods section. Among the 11921 filtered genes, 3733 were differentially expressed between cancer and benign conditions (adjusted p value <0.05). This accounts for 7% of the overall genes included in the array and 31% of the filtered genes. Of these, 1984 were overexpressed in malignant condition. Several of these genes are currently being targeted in therapeutics including TOP2A, VEGFA, AURKA, PARP1, RAS. Seventy-two genes presented more than a two fold increase of the mean value in breast cancer. As expected, a high proportion of these 72 highly overexpressed genes related to cell cycle activation and mitosis, including cyclins (CCNA2, CCNE2, CCNB2), cell division cycle family (CDC2, CKS1B, CKS2, NUF2), kinesin family members (KIF23, NDC80, KIF4A, KIF18A, MKLP2, KIF14) and centromere proteins (CENPF, CENPE).
Finally, the inventors identified 2675 exons (adjusted p value <0.05) overexpressed in malignant condition who were not located within the 1984 filtered genes whose geometric mean values differ between cancer and benign lesions. These 2675 exon-probes were located in 1559 genes. The splice index for the 2675 exon is reported in supplementary table 4. Numbers of these over-expressed exons located within unchanged genes have potential oncogenic properties. As illustration, probe sets for exon 9 casein kinase 1 delta (CSNK1D), exon 5-6 retinoblastoma binding protein 9 (RBBP9) and exon 21-22 Erbb2 interacting protein (ERBB2IP/LAP2) presented a 1.67 (adjusted p=0.005), 1.45 (adjusted p=0.006) and 1.6 (adjusted p=0.0005) fold increased intensity in malignant as compared to benign tumors. At the opposite, the geo mean ratio between cancer and benign tumors for gene expression levels were 1.09 (p=0.95), 1.04 (p=0.61) and 1.00 (p=0.96) for CSNK1D, RBBP9, ERBB2IP/LAP2 respectively. These findings suggest that a significant proportion of genes identified as unchanged may actually contain differentially expressed exons. These considerations suggest that analyses at exon level provide additional information to analyses at gene level, to decipher the transcriptional program of breast malignancy.
Based on the finding that breast cancer and benign tumors present widely different exonic profiles, the inventors developed a molecular classifier for breast cancer diagnosis on FNA samples.
Patients et Methods.
Patients.
94 patients have been included in the present study. These patients referred at the Institut Gustave Roussy for a clinical or radiological breast lesion in 2006. FNA was performed in all of these patients to assess the diagnosis of the breast lesion. A breast adenocarcinoma was diagnosed in 70 patients, and a benign lesion was diagnosed in 24 patients. Breast cancer diagnosis was based on the identification of cancer cells on the CNB or surgical specimen. The diagnosis of benign lesion was based on the identification of non-malignant epithelial cells on CNB and the identification of non-malignant epithelial cells in FNA sample together with lack of radiological change after 3 or 6 months follow-up. Patient characteristics are reported in Table 2.
RNA was extracted using RNEasy kit (Qiagen) from FNA specimen. Total RNA quality and quantity were evaluated using the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer.
Sample amplification and labeling was performed using the WT-Ovation Pico RNA Amplification System and the FL-Ovation cDNA Biotin Module v2 (NuGen, Inc., part #'s 3300-60 and 4200-60) according to manufacturer's instructions.
Standard methods following recommendations of the manufacturer were used to hybridize the samples. A pan-genomic array has been designed to detect 138,636 splicing events. 20,649 genes are analyzed using this array. The overall number of probes included in this array was 6,079,562. The present study was focused on 703,680 Evidenced probes. Once hybridized, the arrays were washed and stained using the FS450-0001 Fluidics protocol prior to scanning using the Affymetrix GeneChip® Scanner 3000 7G. .DAT and .CEL images were visually inspected for anomalies and accurate grid placement. The .CEL files were imported into Partek® Genomics Suite and the data were processed using the Robust Multichip Average (RMA) background correction method followed by GC content background correction. Quantile normalization was performed across all arrays, and then data was Log 2 transformed and mean probe summarization was performed.
Data were processed using the RMA background correction method followed by guanine-cytosine content background correction. Quantile normalization was performed across all arrays, and then data was log 2 transformed and mean probe summarization was performed.
Starting from the 703,680 Evidenced probes, the inventors filtered out probes whose expression varied little across the 94 patient training set as follows: first the inventors floored the expression values at 10 (i.e. all expression values lower than 10 were replaced by 10), and secondly they deleted a probe from the data set if the ratio of the maximum by minimum intensity across the 94-patient set was higher or equal to 5 and if the difference between the maximum and minimum intensity was higher or equal to 100. In total 176,832 probes were retained for analysis after the pre-filtering step.
The point biserial correlation coefficient was calculated for each probe between probe expression and a binary indicator which value was defined by the status of the patient (the indicator took the value 0 in patients who had benign tissue and the value 1 in tissues from patients with tumors) (Kraemer H C: Biserial correlation, in Encyclopedia of Statistical Science, Vol 1. Edited by Kotz S, Read C B. New York, Wiley, 1982, pp 276-280). The probes with the highest absolute correlation are those which are the most likely correlated with the occurrence of a tumor. The inventors used the t-test to test whether the point biserial correlation values were significantly different from 0 (Kraemer HC: Biserial correlation, in Encyclopedia of Statistical Science, Vol 1. Edited by Kotz S, Read C B. New York, Wiley, 1982, pp 276-280). In order to take the multiple testing issues into account they used a very stringent cut-off for statistical significance: 10e-8 (1 out of 100 million).
In order to develop a prediction rule to classify tissues from new patients with unknown status (benign vs. malignant), the inventors chose the nearest centroid prediction rule, which has been intensively studied on data from several microarray studies (S. Michiels, S. Koscielny, C. Hill. Prediction of cancer outcome with microarrays: a multiple random validation strategy. Lancet 2005; 365:488-92). This prediction rule classifies new patients according to the Pearson correlation between the expression of their signature probes and the average profiles in the two categories (the average benign and malignant profiles are defined as the vector of the average expression values of the signature probes in patients with benign and malignant tissues of the 94 patient training set), the predicted category being the one with the highest Pearson correlation. The signature probes were defined as those probes for which the p-value of test of significance of the point biserial correlation coefficient with the benign vs. malignant status was below a particular threshold.
Leave-one-out cross-validation was used to estimate the prediction accuracy of the prediction rule. One sample is left out, and the remaining samples are used to build the prediction rule, which is then used to classify the left-out sample. The entire model-building process was repeated for each leave-one-out training set to provide unbiased estimates of the prediction accuracy (Ntzani E E, Ioannidis J P. Predictive ability of DNA microarrays for cancer outcomes and correlates: an empirical assessment. Lancet 2003; 362: 1439-44; Simon R, Radmacher M D, Dobbin K, McShane L M. Pitfalls in the use of DNA microarray data for diagnostic and prognostic classification. J Natl Cancer Inst 2003; 95: 14-8): correlation coefficients were recalculated, ordered according to their absolute value, the signature probes defined and the nearest centroid prediction rule was constructed. Then, the inventors predicted the outcome of the one sample they left out in the first place based on the highest correlation coefficient it had with the average benign and the malignant profile from the 93 other samples. At the end of the leave-one-out cross-validation procedure, the inventors counted in how many cases the predictions were correct and in how many cases the predictions were incorrect.
The inventors repeated the above performance evaluation procedure based on leave-one-out cross-validation for different p-value thresholds for signature genes in order to find an optimal prediction rule yielding the lowest number of misclassified patients. They tried the following four p-values thresholds to define the signature genes: 10e-11, 10e-10, 10e-09 and 10e-8.
The inventors also provide subsets of 50, 100, 150 and 200 polynucleotide probe sets whose expression can be used to classify the benign and malignant status of tissues of new patients.
The inventors found 1,640 Evidenced polynucleotide probe sets that were significantly associated with malignancy at the stringent p-value cut-off of p<10e-8 (see Table 3). The polynucleotide probe sets with positive correlation are those which have increased expression in malignant tissues, those with negative correlation have increased expression in the benign tissues.
The inventors developed an optimal prediction rule based on the methodology explained above. The preferred p-value threshold for defining signature genes was p<10e-9. This p-value threshold corresponds to the first 1228 polynucleotide probe sets in Table 3 (with identifier no 1 to no 1228). When using leave-one-out cross-validation to estimated the prediction accuracy of the prediction rule with this particular threshold, they obtained 0 out of 24 patients (0%) with benign status that were misclassified and 0 out of 70 patients with malignant status that were misclassified (0%). This corresponds to a sensitivity of 100% and a specificity of 100%.
In order to find subsets of smaller amounts of polynucleotide probe sets to be used for classifying the benign or malignant status of tissues, prediction rules were constructed using the top 50, top 100, top 150 and top 200 polynucleotide probe sets of Table 4 (with identifiers no 1 to no 50, no 1 to no 100, no 1 to no 150 and no 1 to no 200). The estimated prediction accuracies by leave-one-out crossvalidation were as follows:
using the top 50 polynucleotide probe sets: 1 out of 24 patients with benign status were misclassified and 7 out of 70 patients with malignant status.
using the top 100 polynucleotide probe sets: 1 out of 24 patients with benign status were misclassified and 5 out of 70 patients with malignant status.
using the top 150 polynucleotide probe sets: 0 out of 24 patients with benign status were misclassified and 6 out of 70 patients with malignant status.
using the top 200 polynucleotide probe sets: 0 out of 24 patients with benign status were misclassified and 4 out of 70 patients with malignant status.
Table 5 contains the average profiles of the preferred 1,228 signature polynucleotide probe sets with p<10e-9 measured on the 24 benign and 70 malignant tissues of the training set. Thus, the preferred set of probes to be used for predicting the malignant status of a tissue of a future patient is reported here.
An additional 100 patients referred at the Institut Gustave Roussy for a clinical or radiological breast lesion in 2006-2007 for which a FNA was performed to assess the diagnosis of the breast lesion has been analyzed by exon profiling. The prediction rule using the preferred 1228 signature polynucleotide probe sets has been applied to this independent validation set.
The inventors have developed an optimal prediction rule based on the methodology reported previously. The preferred p-value threshold for defining signature genes was p<10e-9. When using leave-one-out cross-validation to estimate the prediction accuracy of the prediction rule with this particular threshold, the inventors accurately classified all the samples as shown in
A validation set was carried-out that included overall 71 samples. Of these, 21 and 50 were benign lesions and breast cancer respectively. Using this dataset, the inventors assessed the performances of the 1228 signature probes to predict for breast cancer as opposed to benign lesions. Overall 68 out of 71 samples were accurately classified (96%). Sensitivity and specificity of the tests were 96% (95% CI: 90.5-100) and 95% (95% CI: 86.1-100). The correlation of each specimen with the malignant and benign profile is reported in
The inventors then evaluated the add-value of molecular predictor to cytological exam, and to a model that included classification of American College of Radiology (ACR) and age. Cytological exam failed to provide a definitive diagnosis in 5 out of 71 patients. Four out of these 5 patients were accurately classified by the molecular predictor. The proportion of explained variation (PEV) by Clinical model (age and ACR) was 75%+−8%, while the PEV by molecular predictor was 81% (+/−10%). When clinical model and molecular predictor were combined, the PEV was 87% (+/−7%), suggesting that molecular predictor presented an add-value of 12% (+/−7%) to the clinical model.
Using the top 50 polynucleotide probe sets: 1 out of 21 samples from patients with benign status were misclassified and 8 out of 50 samples from patients with malignant status.
Using the top 100 polynucleotide probe sets: 1 out of 21 samples from patients with benign status were misclassified and 7 out of 50 samples from patients with malignant status.
Using the top 150 polynucleotide probe sets: 1 out of 21 samples from patients with benign status were misclassified and 10 out of 50 samples from patients with malignant status.
Using the top 200 polynucleotide probe sets: 1 out of 21 samples from patients with benign status were misclassified and 7 out of 50 samples from patients with malignant status.
These data demonstrate that top probe set, even if some misclassifications occur, gives a good prognosis for a breast tumor.
Moreover, the use of the 1228, or 1640, polynucleotide probe sets according to the invention allows, without ambiguity, and misclassification, to determine if a breast tumor is benign or malignant.
In the present study, the inventors have reported the exonic portrait of breast malignancy. The analysis of differential exonic events between malignant and benign lesions provides a complete picture of genomic anomalies associated with breast malignancy. The present study reveals a high level of genomic dysregulation between malignant and benign breast lesions. The inventors found out that 56652 out of 703680 evidenced probes (8%) were different among the two conditions. When the analysis focused on gene expression level (geo mean of probe sets), 3733 out of 20649 genes (7%) were differentially expressed between malignant and benign lesions. Analysis of gene expression levels revealed overexpression of candidate genes for further investigations. Pituitary Tumor Transforming 1 (PTTG1) has been previously reported to be overexpressed in pituitary tumors, thyroid cancer, colon cancer and glioma and to mediate malignant transformation (Vlotides G, Eigler T, Melmed S. Pituitary tumor-transforming gene: physiology and implications for tumorigenesis. Endocr Rev. 2007 April; 28(2):165-86). Neuroepithelial cell transforming gene 1 (NET1) is a guanine exchange factor that has been reported to mediate oncogenic transformation through activation of RhoA (Chan A M, Takai S, Yamada K, Miki T. Isolation of a novel oncogene, NET1, from neuroepithelioma cells by expression cDNA cloning. Oncogene. 1996 Mar. 21; 12(6):1259-66). Pathway analysis revealed that genes involved in spliceosome assemble were enriched in the malignant condition. Spliceosome mediates alternative splicing, a phenomenon involved in oncogenesis (Lønning P E, Knappskog S, Staalesen V, Chrisanthar R, Lillehaug J R. Breast cancer prognostication and prediction in the postgenomic era. Ann Oncol. 2007 August; 18(8):1293-306; Pajares M J, Ezponda T, Catena R, Calvo A, Pio R, Montuenga L M. Alternative splicing: an emerging topic in molecular and clinical oncology. Lancet Oncol. 2007 April; 8(4):349-57). Present study suggests that alternative splicing could significantly contribute to the molecular profile of breast cancer. Based on this consideration, the inventors further evaluated to what extent analyses at exonic level could provide additional information to the ones provided by analyses at gene expression level.
The inventors identified that 2675 exon-probes that did not belong to genes identified as differentially expressed, actually presented a significantly higher intensity in breast cancer. A high proportion of these 2675 exon-probes presented a significantly increased splice index in breast cancer as compared to benign tumors. These exon-probes were considered to present a higher difference of intensity between cancer and benign lesions, as compared to the one of their relative genes. Several of these exons are located within genes linked to cancer biology (casein kinase 1, delta (Gao Z H, Seeling J M, Hill V, Yochum A, Virshup D M. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci USA. 2002 Feb. 5; 99(3):1182-), retinoblastoma binding protein 9 (Woitach J T, Zhang M, Niu C H, Thorgeirsson S S. A retinoblastoma-binding protein that affects cell-cycle control and confers transforming ability. Nat Genet. 1998 August; 19(4):371-4) and ERB2 interacting protein (Jaulin-Bastard F, Arsanto J P, Le Bivic A, Navarro C, Vély F, Saito H, Marchetto S, Hatzfeld M, Santoni M J, Birnbaum D, Borg J P. Interaction between Erbin and a Catenin related protein in epithelial cells. J Biol Chem. 2002 Jan. 25; 277(4):2869-75)). These data point out the limits of analyses at gene expression level to define the transcriptional profile of diseases, and suggest that analysis at exon level could capture biological information that is missed by analysis at gene expression level.
Based on the finding that breast cancer presents a widely different exonic profile as compared to benign tumors, the inventors evaluated whether this technology could be used for breast cancer diagnosis. Pathologic exam of tumor biopsy is the gold standard for cancer diagnosis. Nevertheless, such approach presents several limitations including the need for invasive procedures and, in some tumor types, misdiagnosis. In the present study, the inventors have shown that a large scale molecular analysis based on fine needle aspiration could allow breast cancer diagnosis. This finding opens new ways in the field of cancer diagnosis. Applied to breast cancer, it could allow a more effective post-screening pattern of care by increasing the metric performances of fine needle aspiration. Although biopsy is the current gold standard for breast cancer diagnosis, it is associated with both morbidity and treatment delays (Meunier M, Clough K. Fine needle aspiration cytology versus percutaneous biopsy of nonpalpable breast lesions. Eur J Radiol. 2002 April; 42(1):10-6). Fine needle aspiration is a safer and easier approach but is associated with a high rate of false negative results. In the present study, molecular diagnosis by a 1228-probe signature using FNA samples was associated with 100% accuracy in a training set and 96% accuracy in a validation set. Interestingly, molecular classifier provided accurate diagnosis in 4 out of 5 samples for which the cytological exam was not conclusive. Altogether, these data suggested that the 1228-probe signature could be a complementary tool to cytological exam for the breast cancer diagnosis. This tool could allow avoiding biopsies and unnecessary surgery in patients with benign lesions of the breast. At the opposite, for malignant lesions, this approach could speed-up the diagnosis and could allow decreasing time to surgery. The development of molecular assays for cancer diagnosis could be of special interest in other tumor types. As illustration, the benign nature of thyroid nodules is often difficult to be determined preoperatively, leading to unnecessary surgeries (Poller D N, Stelow E B, Yiangou C. Thyroid FNAC cytology: can we do it better? Cytopathology. 2008 February; 19(1):4-10). A molecular assay could dramatically decrease the need for surgery in this setting.
Overall, the present study reports the full description of exons differentially expressed between breast malignant and benign tumors. These data allowed the identification of candidate genes (PTTG1, NET1) for further functional validation in breast cancer. In addition it suggests that alternative splicing significantly contributes the molecular profile of breast cancer, leading to the identification of exons with a significantly higher splice index in cancer, but located within unchanged gene.
Finally, the splice array technology allowed the development of an exon signature for breast cancer diagnosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08100106.7 | Jan 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/050060 | 1/5/2009 | WO | 00 | 9/29/2010 |
