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WO2014104867A1 - Probes and primers for detection of the her2 gene and a regulator gene (ribosomal) in multiplex format: use in selecting treatment for her2 breast cancer - Google Patents

Probes and primers for detection of the her2 gene and a regulator gene (ribosomal) in multiplex format: use in selecting treatment for her2 breast cancer Download PDF

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WO2014104867A1
WO2014104867A1 PCT/MA2013/000023 MA2013000023W WO2014104867A1 WO 2014104867 A1 WO2014104867 A1 WO 2014104867A1 MA 2013000023 W MA2013000023 W MA 2013000023W WO 2014104867 A1 WO2014104867 A1 WO 2014104867A1
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seq
gene
her2
qpcr
primers
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El Hassane Sefrioui
Abdeladim MOUMEN
Amrani Manale El
Nadia BOUCHOUTROUCH
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Moroccan Foundation For Advanced Science Innovation & Research (mascir)
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Moroccan Foundation For Advanced Science Innovation & Research (mascir)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Probes and primers for detecting the HER2 gene and a (ribosomal) control gene in multiplex format Applications in the choice of HER2 breast cancer treatment
  • the invention recommends novel probes, primers, probe sets, primer sets, probe sets and primers for detecting and quantifying HER2 gene expression and a normalizing control gene using real-time quantitative PCR (qPCR). in a simplex or multiplex format.
  • the control genes selected in this convention (RPL30, RPLP37, and MRPL19) belong to the family of coding genes for ribosomal proteins and are described here for the first time in HER2 quantification.
  • the quantification of the expression of the HER2 gene and gene control by qPCR will select cancer patients overexpressing the HER2 gene and candidates for treatment with anti-HER2 antibody (Herceptin ®).
  • breast cancer is the leading cause of cancer death in women. It is also the most common cancer of women's cancers. In Morocco, breast cancer affects more than 15,000 women every year (Casablanca Regional Cancer Registry, 2004, 2007 edition).
  • the HER2 human epidermal growth factor receptor 2 gene is found on chromosome 17 and encodes the HER2 growth factor receptor, a transmembrane protein with tyrosine kinase activity and overexpressed in 30% of breast cancers. HER2 overexpression induces gene amplification and its activation induces proliferation of tumor cells, stimulation of angiogenesis and metastasis.
  • Herceptin ® is a monoclonal antibody that specifically recognizes the extramembrane domain of the HER2 protein (ECD) and thus inhibits the tumor activity of the latter.
  • ECD extramembrane domain of the HER2 protein
  • IHC immunohistochemistry
  • Herceptin ® is an antibody that detects HER2 protein at the cell membrane level
  • FISH fluorescence in situ hybridization
  • FISH allows to directly visualize the amplification of the HER2 gene using fluorescent DNA probes
  • IHC and FISH mainly lack quantitative and qualitative assays, they are less specific, they use complex antibodies or hybridizations, they take a lot of time (several days), the final score requires several examiners, in addition
  • qPCR real-time PCR
  • qPCR uses in parallel a housekeeping gene that is expressed ubiquitously in all the cells of the body and will allow the exact quantification of the target gene.
  • GAPDH Bosset A
  • beta-actin beta-actin
  • the list of human control genes are cited by Eisenberg et al (Trends in Genetics 2003; 19: 362-
  • Eisenberg et al Terends in Genetics 2003; 19: 362-
  • the use of a control gene whose expression is not stable between the different samples analyzed affects the validity of the results. For this reason, it is necessary to test several control genes and choose the most stable.
  • 3 stable control genes were selected after being validated and confirmed simultaneously by 2 different international statistical software Genorm and GenFinder.
  • the invention relates to the use of a set of new probes and primers and a qPCR method in simplex or multiplexed condition for the detection and quantification of HER2 gene transcripts which thus allows the diagnosis and selection or non Herceptin ® as a basic treatment for breast cancer and / or any other HER2 overexpressing cancer.
  • the method of the present invention represents an improvement of the other prior methods mentioned above and allows a) to gain in reliability and reproducibility by using more stable normalizing control genes b) to gain in sensitivity using newer probes and more specific primers and more sensitive c) a reduction in the cost of consumables and the time of completion because the amplification of the transcripts of the HER2 gene and the control gene are done simultaneously in the same well of qPCR.
  • the detection and quantification of transcripts of the target genes is done by the qPCR method in different steps a) designing and synthesizing novel nucleotide sequences of probes and primers that hybridize
  • the control gene is selected from the group RPL30, MRPL19, RPLP37, and any other control genes encoding ribosomal proteins c) quantification of the copy number of the HER2 gene on the chromosome D) quantification of the number of copies of the control gene e) comparison of the number of HER2 gene copies with that of the control gene by obtaining a ratio of the number of copies between the HER2 gene and the control gene (f) the evolution of this gene ratio between cancer patients with IHC scores 3+, 2+ or 0 determines a salt; he minimum detection and identification of candidate patients for treatment with Herceptin ®.
  • a) the primers allowing the detection of the transcript
  • HER2 in a sample test have oligonucleotide sequences selected from a group of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, b) the primers to detect the transcript of the control gene RLP30 for example in a sample test have oligonucleotide sequences selected from a group of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.
  • the probe for detecting the HER2 gene transcript in a sample test has a sequence selected from: SEQ ID: 9, SEQ ID: 12.
  • the probe for detecting the transcript of the RLP30 control gene in a sample test has a sequence selected from SEQ ID: 10, SEQ ID: 11.
  • all primers that amplify the HER2 or RLP30 gene transcripts in a sample test include:
  • a "forward" primer having a sequence selected from the group: SEQ ID NO: 1, SEQ ID NO: 3, and a "reverse” primer having a sequence selected from SEQ ID NO: 2, QEQ ID NO : 4
  • RLP30 for example, a "forward" primer having a sequence from the group: SEQ ID NO: 5, SEQ ID NO: 7, and a "reverse” primer having a sequence selected from SEQ ID NO: 6, SEQ ID NO: 8.
  • the present invention also relates to a method for the detection of HER2 and RPL30 gene transcripts in the same sample test. This method takes place in several stages:
  • HER2 For HER2, contact a sample test with a "forward" primer having a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3, and a "reverse” primer selected from SEQ ID NO: 2 or SEQ ID NO: 4 under specific amplification conditions to generate a target sequence.
  • the probe has a sequence selected from SEQ ID: 12 and SEQ. ID NO: 9 and for RLP30, the probe has a sequence selected from SEQ ID NO: 10 and SEQ ID NO: 11.
  • the amplification of a gene consists; to put the test sample in an amplification reaction in the presence of amplification reagents.
  • the amplification reaction may be PCR, RT-PCR or qPCR.
  • a probe contains a fluorescent unit (reporter) attached to the 5 'region of DNA.
  • a probe may contain a quencher portion at the 3 'region of DNA. Quantification of the fluorescence unit allows quantification of the target gene (HER2 or RLP30).
  • the amplification of the HER2 and RLP30 gene transcripts by the qPCR of the present invention can be in simplex or multiplex format.
  • FIG. 1 Graph showing PCR cycles versus Delta Rn for HER2 gene transcripts.
  • the HER2 positive breast cancer cell line (SKBR3) is used as the source of the genetic material undergoing qPCR of the present invention.
  • 7 concentrations of cDNA DNA resulting from the reverse transcription of all of the SKBR3 mRNAs (4 ⁇ g-25 ng) were used.
  • Figure 2 Standard curve (Cycle threshold (Ct) -versus-log amount cDNA) reconstituted by the results obtained in Figure 1.
  • Table 1 Ct values used in the standard curve presented in Figure 2.
  • Figure 3 Curve representing cycles of PCR as a function of Delta Rn for HER2 gene transcripts. Two samples of breast cancer patients (score 3+) and 2 others with a negative score (0) were tested using the qPCR of the present invention.
  • Figure 5 Curves of the most stable and unstable control genes in biopsies of HER2 breast cancer patients. The expression of several control genes was quantified by the qPCR of the present invention.
  • the invention provides probes, primers, primer sets, probe sets, and primers that can be used to amplify, detect, and quantify the HER2 gene or control gene in a test sample.
  • the invention also advocates a method for detecting HER2 gene transcripts and the RPL30 control gene in a test sample using the sets of primers and probes cited above.
  • the sets of primers and probes of the present invention allow better sensitivity and specificity for detecting HER2 and RPL30.
  • the amplification of the transcripts of the HER2 gene and of the control gene is at least qPCR in simplex or multiplexed format thus allowing a better quantification of HER2 and a gain in time and cost.
  • the more sensitive and specific quantification of HER2 patients will select candidates for treatment with Herceptin ®.
  • the word 'HER2' described in the present invention represents the gene 'human epidermal growth factor 2' which codes for the HER2 protein, a thyrosine kinase which can induce metastasis and tumor proliferation, in case of overexpression.
  • Detection and quantification of the HER2 gene transcript can support the diagnosis and select candidates for Herceptin ® treatment.
  • the word 'gene' used in the present invention refers to a nucleic acid sequence of the DNA molecule occupying a specific region in a chromosome and permits the encoding of instructions for RNA synthesis.
  • Oligonucleotide' is a sequence composed of DNA or RNA or their combination with a length that ranges from 10 to 70 nucleotides. Oligonucleotides can be synthesized by several methods but not limited to that of 5'-3 'synthesis based on the use of beta-octanoethyl phosphate protecting groups (Rosenthal et al., Terahedron Letter 24: 1691, 1983; Nude Acids Res 4: 1135, 1977) or the phosphochloridite method (Matteucci J AM CHEM SOC 103: 3185-91, 1981).
  • Amplification as used herein refers to one or more methods capable of copying a nucleic acid thereby allowing an increase in the number of copies of a target nucleic acid sequence.
  • the amplified sequence can be a ribonucleotide acid (RNA) or a deoxyribonucleotide acid (DNA)
  • RNA ribonucleotide acid
  • DNA deoxyribonucleotide acid
  • the word 'reverse transcription' mentioned here represents a method for the synthesis of complementary DNA (cDNA) from RNA molecule.
  • the cDNA will be used in the PCR, RT-PCR or qPCR method.
  • the word "primer” refers to a sequence of oligonucleotides synthesized chemically or naturally.
  • the primer is the point of initiation of DNA synthesis under optimal temperature conditions and in the presence of specific enzyme, buffers, nucleotides and complementary nucleotide sequences.
  • the word 'probe' referred to herein refers to a nucleotide sequence that forms a hybrid structure with a target sequence in a molecule of a test sample.
  • the word 'PCR' refers to a method from which a cDNA or DNA sample is added to a solution in the presence of unattached nucleotides (eg dNTPs), 2 oligonucleotide primers (forward and reverse); and the DNA polymerase enzyme (preferably heat-resistant Taq polymerase) that catalyzes the formation of DNA from as early as 2 primers and dNTPs.
  • the mixed solution is heated to 94-96 ° C to denature the DNA molecule and form 2 single strands of DNA.
  • the primers will then bind specifically to the single-stranded DNA thereby allowing the DNA polymerase to catalyze the attachment of the dNTPs to the primers.
  • the word 'RT-PCR' reverse transcriptase-PCR used in the present invention represents a PCR method whose starting material is not directly DNA but an RNA translated into cDNA after reverse transcription.
  • qPCR quantitative PCR or real time RT-PCR
  • the word "qPCR” represents a PCR method for studying the products of the PCR reaction during the first DNA amplification steps.
  • the PCR products are detected by probes labeled at the 5 'end by a transmitter fluorochrome (reporter), and at their 3' end by a fluorescent or non-fluorescent quencher, which inhibits the emission of the reporter when they are nearby.
  • the signal intensity of the fluorescence measured during the amplification reaction is proportional to the number of newly formed products (amplicons).
  • the measurement in DNA or cDNA is in logarithm.
  • threshold cycle Cycle Threshold
  • the time of appearance of this threshold signal is dependent on the amount of matrix initially present in the amplified sample.
  • the calculated Ct is inversely proportional to the decimal logarithm of the number of initial copies.
  • probes in qPCR are the meecular beacons and TaqMan probes that use the fluorogenic exonuclease activity of the Taq polymerase enzyme to measure the amount of the DNA sequence in a test sample.
  • the qPCR of the present invention uses the TaqMan probes and the amplification analysis is made by ABI PRISM 7900HT 'sequence detection system' which is a screening system that can detect and quantify nucleic acids.
  • ABI PRISM 7900HT 'sequence detection system' which is a screening system that can detect and quantify nucleic acids.
  • the probe reporter may be FAM, JOE, YAKYE ( ⁇ ) and BBQ quencher, BI-iQl, or TAMRA.
  • the simplex word of the present invention represents a test that does not run concurrently with other tests.
  • simplex qPCR reflects the detection of copy number of a single gene in a reaction tube.
  • gene control or "housekeeping gene” described in the present invention represents a gene generally similarly expressed by all cells of an organism.
  • control genes cited in the literature beta-actin, glyceraldehyd-3-phosphate dehydrogenase (GAPDH), ABL1 and others are found.
  • the multiplex word cited in the present invention refers to a test that takes place ⁇
  • the multiplexed qPCR recommends a detection of the number of copies of at least 2 genes in the same reaction tube.
  • the 2 genes HER2 and RPL30 can be simultaneously detected by qPCR.
  • test sample refers to a fresh or waxed tumor biopsy sample, primary cells, cell lines, saliva or other organic fluids expressing or HER2 overexpressing.
  • the test sample can be of human or animal origin expressing or overexpressing the HER2 gene that can be amplified using the primers and probes of the present invention.
  • the qPCR method cited in the present invention makes it possible to determine the number of copies of the HER2 gene in a test sample by quantifying the number of HER2 copies relating to a control gene.
  • the control gene is selected from the genes RLP30, RP37, MPRL19 and any control gene coding for ribosomal proteins.
  • Example 1 represents a description of the protocol used in the qPCR method of the present invention. The steps of RNA extraction, cDNA synthesis and qPCR are described in this protocol.
  • Example 2 Figure 1 and Figure 2 and Table 1 describe the high sensitivity and specificity of the qPCR of the present invention for detecting HER2 gene transcripts at the HER2-overexpressing SKBR3 breast cancer human line.
  • the results of the qPCR of the present invention are shown in Figure 1 (amplification curve: cycle-vs-Delta Rn), Figure 2 (standard curve: amount of SKBR3 cDNA vs Ct-values) and Table 1 ( value of Ct corresponding to the standard curve).
  • the quality values obtained are according to international IVD standards (R2> 0.95, slope between -3.0 and 3.9 and efficiency of 90-112)) with a standard curve with a slope of -3.08, an R2 of 0.98 and an efficiency. of 111 ( Figure 2).
  • the amplification curve ( Figure 1) also shows a perfect pace.
  • Example 3 shows the amplification curve (cycle-vs-Delta Rn) of the qPCR of the present invention for detecting the HER2 gene transcript in HER2-overexpressing breast cancer patients (score 3+ ) (green curve) or expressing normally HER2 (score 0) (blue curve) ( Figure 3). Patients with the score 3+ show a low Ct value and thus a higher amount of HER2 compared to patients with score 0 ( Figure 3).
  • Example 4 shows the amplification curve (cycle-vs-Delta Rn) of the qPCR of the present invention for detecting the HER2 gene transcript in HER2-overexpressing breast cancer patients (score 3+ ) (green curve) or expressing normally HER2 (score 0) (blue curve) ( Figure 3). Patients with the score 3+ show a low Ct value and thus a higher amount of HER2 compared to patients with score 0 ( Figure 3).
  • Example 4 shows the amplification curve (cycle-vs-Delta
  • Figure 4a shows the Ct (triplicata) values of the qPCR of the present invention in breast cancer patients with a score of 3+, 2+ or 0 ( Figure 4).
  • the Ct values of the present qPCR correspond well to the values of the IHC. Indeed, for the score of 3+, the Ct values are lower (31-33.5). In the case of patients with a negative score, the Ct values are between 34 and 38. Then the over-expressing HER2 SKBR3 line has a Ct value of 27.2 which is closer to 3+.
  • Figure 4b shows the results of qPCR in the form of 2deltadeltaCt values which is the HER2 overexpression index number by contribution to the negative cell line for HER2. For normalization, the RPL37 control gene was used.
  • FIG. 5 shows the curve of the most stable control genes. Measurement of the stability of the expression of several control genes by qPCR on biopsies of HER2 breast cancer patients was validated using the Genorm software. The control genes RPL30, RPLP37 and MRPL19 are all to the right of the curve in the part of the most stable genes ( Figure 5). The same control genes were also identified as the most stable in breast cancer cell lines (SKBR3, MCF7, DA) using the same Genorm software (results are not shown).
  • Example 1 qPCR protocol of the present invention for the quantification of HER2 at the HER2 overexpressing SKBR3 cell line or paraffinic breast cancer biopsies !
  • RNA extraction from breast cancer patient biopsy samples was done by the Qiagen kit (QiagenRNAeasy FFPE extraction kit) or that of AMSBIO (England) following the recommendations of the suppliers.
  • the quality and purity of the RNA obtained is tested by agarose gel electrophoresis, or by the Bioanalyzer (Agilant).
  • the complementary DNA (cDNA) is obtained from total RNA extracted from the test sample.
  • the cDNA synthesis is carried out using the Thermocycler (Applied Bios stems) using the RNA kit to cDNA Reverse Transcription according to the supplier's recommendations (Applied Biosystems).
  • the primers and the probe of the target genes and 5 ⁇ of cDNA are added to the mastermix of the qPCR reaction (TaqMan Fast Universal Master PCR mix: Applied Biosystems) for a final volume of 25 ⁇ .
  • step 1 at 95 ° C for 20 seconds
  • step 2 cycle 50
  • step 3 at 60 ° C for 30 seconds.
  • Examples 2, 3, 4a, 4b The performance of QPCR of the present invention for quantifying HER2 in the SKBR3 breast cancer line and in HER2 breast cancer patients (amplification curve, standard curve, Ct values and 2deltadeltaCt)
  • Reverse primer 5 'Gtccttatagtgggcacagg 3' (SEQ ID No. 2)

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Abstract

The invention relates to novel probes, primers, sets of probes, set of primers, and sets of probes and primers for detecting and quantifying the expression of the HER2 gene and a normalising regulator gene, using quantitative PCR (qPCR) in real time in a simplex or multiplex format. The selected regulator genes (RPL30, RPLP37, and MRPL19) belong the family of genes coding for ribosomal proteins and are described here for the first time in relation to HER2 quantification.

Description

Sondes et amorces pour détecter le gène HER2 et un gène (ribosomale) dé contrôle en format multiplex : Applications dans le choix de traitement du cancer de sein HER2  Probes and primers for detecting the HER2 gene and a (ribosomal) control gene in multiplex format: Applications in the choice of HER2 breast cancer treatment

DOMAINE TECHNIQUE TECHNICAL AREA

L'invention préconise de nouvelles sondes, amorces, sets de sondes, sets d'amorces, sets de sondes et amorces pour détecter et quantifier l'expression du gène HER2 et un gène contrôle normalisateur en utilisant la PCR quantitative en temps réel (qPCR) sous un format simplexe ou multiplexe. Les gènes contrôles sélectionnés dans la présente convention (RPL30, RPLP37, et MRPL19) appartiennent à la famille des gènes codants pour des protéines ribosomales et sont décrits ici pour la première fois dans la quantification de HER2. The invention recommends novel probes, primers, probe sets, primer sets, probe sets and primers for detecting and quantifying HER2 gene expression and a normalizing control gene using real-time quantitative PCR (qPCR). in a simplex or multiplex format. The control genes selected in this convention (RPL30, RPLP37, and MRPL19) belong to the family of coding genes for ribosomal proteins and are described here for the first time in HER2 quantification.

En accord avec cette invention, la quantification de l'expression du gène HER2 et le gène contrôle par qPCR permettra de sélectionner les patients cancéreux, surexprimant le gène HER2 et candidats pour le traitement avec l'anticorps anti-HER2 (Herceptin®). In accordance with this invention, the quantification of the expression of the HER2 gene and gene control by qPCR will select cancer patients overexpressing the HER2 gene and candidates for treatment with anti-HER2 antibody (Herceptin ®).

ETAT DE LA TECHNIQUE ANTERIEURE STATE OF THE PRIOR ART

Le cancer du sein représente la première cause de mortalité par cancer chez la femme. Il est aussi le cancer le plus répandu des cancers de la femme. Au Maroc, le cancer du sein frappe plus de 15 000 femmes chaque année (registre des cancers Région Casablanca, 2004, édition 2007).  Breast cancer is the leading cause of cancer death in women. It is also the most common cancer of women's cancers. In Morocco, breast cancer affects more than 15,000 women every year (Casablanca Regional Cancer Registry, 2004, 2007 edition).

Le gène HER2 (human epidermal growth factor receptor 2) se trouve sur le chromosome 17 et code pour le récepteur de facteur de croissance HER2, une protéine transmembranaire ayant une activité tyrosine kinase et étant surexprimée dans 30% des cancers de sein. La surexpression de HER2 induit une amplification génique et son activation induit la prolifération des cellules tumorales, la stimulation de l'angiogénèse et la métastase. The HER2 (human epidermal growth factor receptor 2) gene is found on chromosome 17 and encodes the HER2 growth factor receptor, a transmembrane protein with tyrosine kinase activity and overexpressed in 30% of breast cancers. HER2 overexpression induces gene amplification and its activation induces proliferation of tumor cells, stimulation of angiogenesis and metastasis.

L'Herceptin® est un anticorps monoclonal qui reconnaît spécifiquement le domaine extra- membranaire de la protéine HER2 (ECD) et inhibe ainsi l'activité tumorale de cette dernière. Plusieurs essais cliniques randomisés ont démontré un bénéfice en survie globale de l'Herceptin® en combinaison avec la chimiothérapie chez les patientes atteints par un du cancer de sein surexprimant HER2 (Romond EH ; N Engl J Med. 2005; 353:1673-684. Gianni L ; Lancet Oncol ; 2011;12:236-244]. D'autre essai cliniques ont montré la même efficacité de l'Herceptin® dans le cas du cancer de l'estomac ou de l'œsophage surexprimant HER2 (Bang YJ ; Lancet. 2010; 16;376(9749):1302) La méthode standard actuelle permettant de quantifier relativement l'expression de HER2 d'une tumeur est l'immunohistochimie (IHC). Le but de l'IHC est de mettre en évidence une surexpression de la protéine HER2, qui se caractérise par un score HER2 (3+), lequel indique un traitement par l'Herceptin® (Genentech, San Francisco, Calif.). L'Herceptin® est un anticorps qui détecte la protéine HER2 au niveau de la membrane cellulaire. La technique de FISH (fluorescence in situ hybridization) est utilisée dans les situations de scores intermédiaires (Her2 (2+)). La FISH permet de visualiser directement l'amplification du gène HER2 en utilisant des sondes ADN fluorescentes. Cependant, l'IHC et la FISH manquent surtout d'essais quantitatifs et qualitatifs, elles sont moins spécifiques, utilisent des anticorps ou des hybridations complexes, prennent énormément de temps (plusieurs jours), le score final nécessite plusieurs examinateurs, en plus la méthode FISH est très coûteuse. Herceptin ® is a monoclonal antibody that specifically recognizes the extramembrane domain of the HER2 protein (ECD) and thus inhibits the tumor activity of the latter. Several randomized clinical trials have demonstrated a benefit in overall survival of Herceptin ® in combination with chemotherapy in patients with HER2 overexpressing breast cancer (Romond EH; N Engl J Med 2005; 353: 1673-684; Gianni L; Lancet Oncol; 2011; 12: 236-244). Other clinical trials have shown the same efficacy of Herceptin ® for HER2-overexpressing gastric or esophagus cancer (Bang YJ, Lancet 2010; 16; 376 (9749): 1302) The current standard method for relatively quantifying HER2 expression a tumor is immunohistochemistry (IHC) The goal of IHC is to demonstrate an overexpression of the HER2 protein, which is characterized by a HER2 (3+) score, which indicates treatment with Herceptin ® ( Genentech, San Francisco, Calif.) Herceptin ® is an antibody that detects HER2 protein at the cell membrane level, and FISH (fluorescence in situ hybridization) is used in intermediate-score situations (Her2 (2 +)) FISH allows to directly visualize the amplification of the HER2 gene using fluorescent DNA probes However, IHC and FISH mainly lack quantitative and qualitative assays, they are less specific, they use complex antibodies or hybridizations, they take a lot of time (several days), the final score requires several examiners, in addition the FISH method is very expensive.

C'est pour les raisons citées ci-dessus qu'il est préférable d'identifier des méthodes de quantification de HER2 avec plus de sensibilité et de fiabilité, de meilleures reproductibilités quantitatives et qualitatives et avec un coût plus bas. It is for the reasons mentioned above that it is preferable to identify HER2 quantification methods with greater sensitivity and reliability, better quantitative and qualitative reproducibility, and lower cost.

Actuellement, la PCR en temps réel (qPCR) qui consiste en une amplification du gène cible a été appliquée avec succès en diagnostics cliniques (Hughes TP ; N ; Engl J Med. 2003;349:1423-1432) en raison de sa haute spécificité, de sa rapidité, de sa fiabilité et de son faible cout. Currently, real-time PCR (qPCR) which consists of amplification of the target gene has been successfully applied in clinical diagnoses (Hughes TP; N; Engl J Med 2003; 349: 1423-1432) due to its high specificity , its speed, reliability and low cost.

Pour la quantification d'un gène cible, la qPCR utilise en parallèle un gène contrôle 'housekeeping gene" exprimé de manière ubiquitaire dans toutes les cellules du corps et qui permettra la quantification exacte du gène cible. Dans le cas de la quantification de HER2, plusieurs gènes contrôles ont été utilisés en littérature comme GAPDH (Bofin A, Am J Clin Pathol 2004;122:110-119) et beta-actine (Kao J, PLoS ONE 2009 ; 4 ; e6146)). La liste des gènes contrôles humain sont cités par Eisenberg et al (Trends in Genetics 2003 ; 19 : 362- Cependant, l'utilisation d'un gène contrôle dont l'expression n'est pas stable entre les différents échantillons analysés affecte la validité des résultats. C'est pour cette raison, il faut tester plusieurs gènes contrôles et choisir le plus stable. Dans le cas de la présente invention, 3 gènes contrôles stables ont été sélectionnés après être validés et confirmés simultanément par 2 différents logiciels statistiques internationaux Genorm et GenFinder. For the quantification of a target gene, qPCR uses in parallel a housekeeping gene that is expressed ubiquitously in all the cells of the body and will allow the exact quantification of the target gene.In the case of the quantification of HER2, several control genes have been used in literature such as GAPDH (Bofin A, Am J Clin Pathol 2004; 122: 110-119) and beta-actin (Kao J, PLoS ONE 2009; 4; e6146). The list of human control genes are cited by Eisenberg et al (Trends in Genetics 2003; 19: 362- However, the use of a control gene whose expression is not stable between the different samples analyzed affects the validity of the results. For this reason, it is necessary to test several control genes and choose the most stable. In the case of the present invention, 3 stable control genes were selected after being validated and confirmed simultaneously by 2 different international statistical software Genorm and GenFinder.

EXPOSE DE L'INVENTION SUMMARY OF THE INVENTION

L'invention concerne l'utilisation d'un ensemble de sondes et d'amorces nouveaux et d'une méthode de qPCR en condition simplexe ou multiplexe pour la détection et la quantification des transcrits du gène HER2 qui permet ainsi le diagnostique et le choix ou non de l'Herceptin® comme traitement de base au cancer de sein et/ou tout autre cancer surexprimant HER2. The invention relates to the use of a set of new probes and primers and a qPCR method in simplex or multiplexed condition for the detection and quantification of HER2 gene transcripts which thus allows the diagnosis and selection or non Herceptin ® as a basic treatment for breast cancer and / or any other HER2 overexpressing cancer.

Le procédé de la présente invention représente une amélioration des autres méthodes antérieurs citées ci-dessus et permet a) de gagner en fiabilité et reproductibilité en utilisant des gènes contrôles normalisateurs plus stables b) de gagner en sensibilité en utilisant des nouvelles sondes et amorces plus spécifiques et plus sensibles c) une réduction du coût de consommables et du temps de réalisation du fait que l'amplification des transcrits du gène HER2 et du gène contrôle se font simultanément dans le même puits de qPCR. Selon la présente invention, la détection et la quantification des transcrits des gènes cibles se font par la méthode qPCR en différentes étapes a) concevoir et synthétiser des nouvelles séquences nucléotidique de sondes et amorces qui s'hybrident  The method of the present invention represents an improvement of the other prior methods mentioned above and allows a) to gain in reliability and reproducibility by using more stable normalizing control genes b) to gain in sensitivity using newer probes and more specific primers and more sensitive c) a reduction in the cost of consumables and the time of completion because the amplification of the transcripts of the HER2 gene and the control gene are done simultaneously in the same well of qPCR. According to the present invention, the detection and quantification of transcripts of the target genes is done by the qPCR method in different steps a) designing and synthesizing novel nucleotide sequences of probes and primers that hybridize

spécifiquement sur les transcrits du gène HER2 ou le gène contrôle b) le gène contrôle est sélectionné parmi le group RPL30, MRPL19, RPLP37, et tout autres gènes contrôles codant pour des protéines ribosomales c) quantification du nombre de copies du gène HER2 sur le chromosome 17 d) quantification du nombre de copies du gène contrôle e) comparaison du nombre de copie de gène HER2 à celui du gène contrôle en obtenant un rapport de nombre de copie entre le gène HER2 et le gène contrôle (f) l'évolution de ce rapport entre patients de cancer avec des scores d'IHC de 3+, 2+ ou 0 permet de déterminer un seljil minimum de détection et une identification de patients candidats pour un traitement à l'Herceptin®. Selon une caractéristique de l'invention, a) les amorces permettant la détection du transcritspecifically on the HER2 gene transcripts or the control gene b) the control gene is selected from the group RPL30, MRPL19, RPLP37, and any other control genes encoding ribosomal proteins c) quantification of the copy number of the HER2 gene on the chromosome D) quantification of the number of copies of the control gene e) comparison of the number of HER2 gene copies with that of the control gene by obtaining a ratio of the number of copies between the HER2 gene and the control gene (f) the evolution of this gene ratio between cancer patients with IHC scores 3+, 2+ or 0 determines a salt; he minimum detection and identification of candidate patients for treatment with Herceptin ®. According to one characteristic of the invention, a) the primers allowing the detection of the transcript

HER2 dans un test échantillon ont des séquences d'oligonucleotides sélectionnées parmi un groupe de : SEQ ID NO :1, SEQ ID NO :2, SEQ ID NO :3, et SEQ ID NO :4, b) lés amorces pour détecter le transcrit du gène contrôle RLP30 par exemple dans un test échantillon ont des séquences d'oligonucleotides sélectionnées parmi un groupe de : SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8. HER2 in a sample test have oligonucleotide sequences selected from a group of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, b) the primers to detect the transcript of the control gene RLP30 for example in a sample test have oligonucleotide sequences selected from a group of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.

Selon une autre caractéristique de la présente invention, According to another characteristic of the present invention,

a) la sonde pour détecter le transcrit du gène HER2 dans un test échantillon a une séquence selectionée parmi: SEQ ID : 9, SEQ ID° : 12.  a) the probe for detecting the HER2 gene transcript in a sample test has a sequence selected from: SEQ ID: 9, SEQ ID: 12.

b) la sonde pour détecter le transcrit du gène contrôle RLP30 dans un test échantillon a une séquence sélectionnée parmi SEQ ID : 10, SEQ ID : 11.  b) the probe for detecting the transcript of the RLP30 control gene in a sample test has a sequence selected from SEQ ID: 10, SEQ ID: 11.

En accord avec cette invention, l'ensemble des amorces qui amplifient les transcrit du gène HER2 ou RLP30 dans un test échantillon inclus : In accordance with this invention, all primers that amplify the HER2 or RLP30 gene transcripts in a sample test include:

a) pour HER2, une amorce «forward» ayant une séquence sélectionnée parmi le groupe : SEQ ID NO : 1, SEQ ID NO : 3, et une amorce «reverse» ayant une séquence sélectionnée parmi SEQ ID NO : 2, QEQ ID NO : 4  a) for HER2, a "forward" primer having a sequence selected from the group: SEQ ID NO: 1, SEQ ID NO: 3, and a "reverse" primer having a sequence selected from SEQ ID NO: 2, QEQ ID NO : 4

b) pour RLP30 par exemple, une amorce «forward» ayant une séquence parmi le group : SEQ ID NO : 5, SEQ ID NO : 7, et une amorce «reverse» ayant une séquence sélectionnée parmi SEQ ID NO : 6, SEQ ID NO : 8.  b) for RLP30 for example, a "forward" primer having a sequence from the group: SEQ ID NO: 5, SEQ ID NO: 7, and a "reverse" primer having a sequence selected from SEQ ID NO: 6, SEQ ID NO: 8.

La présente invention concerne aussi une méthode pour la détection des transcrits des gènes HER2 et RPL30 dans un même test échantillon. Cette méthode se déroule en plusieurs étapes : The present invention also relates to a method for the detection of HER2 and RPL30 gene transcripts in the same sample test. This method takes place in several stages:

a) Pour HER2, mettre en contact un test échantillon avec une amorce «forward» ayant une séquence sélectionnée parmi SEQ ID NO : 1 ou SEQ ID NO : 3, et une amorce «reverse» sélectionnée parmi SEQ ID NO : 2 ou SEQ ID NO : 4 dans des conditions d'amplification spécifiques pour générer une séquence cible.  a) For HER2, contact a sample test with a "forward" primer having a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3, and a "reverse" primer selected from SEQ ID NO: 2 or SEQ ID NO: 4 under specific amplification conditions to generate a target sequence.

b) Pour RLP30, mettre en contact un test échantillon avec une amorce «forward» ayant une séquence sélectionnée parmi SEQ ID NO : 5 ou SEQ ID NO : 7, et une amorce reverse sélectionnée parmi SEQ ID NO : 6 ou SEQ ID NO : 8 dans des conditions d'amplification spécifiques pour générer une séquence cible b) For RLP30, contacting a sample test with a "forward" primer having a sequence selected from SEQ ID NO: 5 or SEQ ID NO: 7, and a primer reverse selected from SEQ ID NO: 6 or SEQ ID NO: 8 under specific amplification conditions to generate a target sequence

c) Détecter l'amplification des deux séquences cibles, HER2 et RPL30, dans un même test échantillon avec deux sondes différentes. Pour HER2, la sonde a une séquence Sélectionnée parmi SEQ ID° : 12 et SEQ. ID NO : 9 et pour RLP30, la sonde a une séquence sélectionnée parmi SEQ ID NO : 10 et SEQ ID NO : 11.  c) Detect the amplification of the two target sequences, HER2 and RPL30, in the same sample test with two different probes. For HER2, the probe has a sequence selected from SEQ ID: 12 and SEQ. ID NO: 9 and for RLP30, the probe has a sequence selected from SEQ ID NO: 10 and SEQ ID NO: 11.

Selon la méthode de la présente invention, l'amplification d'un gène consiste; à mettre le test échantillon dans une réaction d'amplification en présence de réactifs d'amplification. La réaction d'amplification peut être une PCR, RT-PCR ou qPCR.  According to the method of the present invention, the amplification of a gene consists; to put the test sample in an amplification reaction in the presence of amplification reagents. The amplification reaction may be PCR, RT-PCR or qPCR.

En accordance avec la présente invention, une sonde contient une unité fluorescente (reporter) attachée à la région 5' d'ADN. En plus, une sonde peut contenir une partie quencher à la région 3' d'ADN. La quantification de l'unité fluorescence permet la quantification du gène cible (HER2 ou RLP30). In accordance with the present invention, a probe contains a fluorescent unit (reporter) attached to the 5 'region of DNA. In addition, a probe may contain a quencher portion at the 3 'region of DNA. Quantification of the fluorescence unit allows quantification of the target gene (HER2 or RLP30).

En accordance aussi avec cette invention, l'amplification des transcrits des gènes HER2 et RLP30 par la qPCR de la présente invention peuvent se faire en format simplexe ou multiplexe.  Also according to this invention, the amplification of the HER2 and RLP30 gene transcripts by the qPCR of the present invention can be in simplex or multiplex format.

BREVE DESCRIPTION DES FIGURES ET TABLEAUX Figure 1 : Graphique représentant les cycles de la PCR en fonction du Delta Rn pour les transcrits du gène HER2. La lignée cellulaire du cancer de sein positive pour HER2 (SKBR3) est utilisée comme source du matériel génétique subissant une qPCR de la présente invention. 7 concentrations d'ADNc (ADN résultant de la transcription inverse de l'ensemble des ARNm du SKBR3) (4pg-25ng) ont été utilisées. BRIEF DESCRIPTION OF THE FIGURES AND TABLES FIG. 1: Graph showing PCR cycles versus Delta Rn for HER2 gene transcripts. The HER2 positive breast cancer cell line (SKBR3) is used as the source of the genetic material undergoing qPCR of the present invention. 7 concentrations of cDNA (DNA resulting from the reverse transcription of all of the SKBR3 mRNAs) (4 μg-25 ng) were used.

Figure 2 : Courbe standard (Cycle threshold (Ct)-versus-log quantité ADNc) reconstituée par les résultats obtenus en Figure 1. Figure 2: Standard curve (Cycle threshold (Ct) -versus-log amount cDNA) reconstituted by the results obtained in Figure 1.

Tableau 1 : Valeurs Ct utilisées dans la courbe standard présentée dans la Figure 2. Figure 3 : Courbe représentant les cycles de la PCR en fonction du Delta Rn pour les transcrits du gène HER2. Deux échantillons de patientes de cancer de sein (score 3+) et 2 autres avec un score négatif (0) ont été testés en utilisant la qPCR de la présente invention. Figure 4a: Courbe des valeurs Ct de la qPCR de la présente invention chez des patientes de cancer de sein avec un score de 3+ (n=2), 2+ (n=2) ou un score négatif (0) (n=4). Table 1: Ct values used in the standard curve presented in Figure 2. Figure 3: Curve representing cycles of PCR as a function of Delta Rn for HER2 gene transcripts. Two samples of breast cancer patients (score 3+) and 2 others with a negative score (0) were tested using the qPCR of the present invention. Figure 4a: Curve of the qtc values of qPCR of the present invention in breast cancer patients with a score of 3+ (n = 2), 2+ (n = 2) or a negative score (0) (n = 4).

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Figure 4b : Courbe des valeurs 2deltadeltaCt de la qPCR de la présente invention chez des patientes de cancer de sein sur-exprimant HER2 par la méthode classique FISH (n=ll).  Figure 4b: Curve of the 2deltadeltaCt values of the qPCR of the present invention in HER2 overexpressing breast cancer patients by the conventional FISH method (n = 11).

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Figure 5 : Courbes des gènes contrôles les plus stables et les moins stables dans les biopsies de patientes de cancer de sein HER2. L'expression de plusieurs gènes contrôles a été quantifiée par la qPCR de la présente invention.  Figure 5: Curves of the most stable and unstable control genes in biopsies of HER2 breast cancer patients. The expression of several control genes was quantified by the qPCR of the present invention.

EXPOSE DETAILLE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION

L'invention préconise des sondes, amorces, ensemble d'amorces, ensemble de sondes et amorces qui peuvent être utilisés pour amplifier, détecter et quantifier le gène HER2 ou le gène contrôle dans un échantillon test. L'invention préconise aussi une méthode de détection des transcrits du gène HER2 et du gène contrôle RPL30 dans un échantillon test en utilisant les ensembles d'amorces et sondes cités ci-dessus. Les ensembles d'amorces et sondes de la présente invention permettent une meilleur sensibilité et spécificité pour détecter HER2 et RPL30. Selon une caractéristique de la présente invention, l'amplification des transcrits du gène HER2 et du gène contrôle se fait au moins en qPCR en format simplexe ou multiplexe permettant ainsi une meilleure quantification de HER2 et un gain en temps et en cout. The invention provides probes, primers, primer sets, probe sets, and primers that can be used to amplify, detect, and quantify the HER2 gene or control gene in a test sample. The invention also advocates a method for detecting HER2 gene transcripts and the RPL30 control gene in a test sample using the sets of primers and probes cited above. The sets of primers and probes of the present invention allow better sensitivity and specificity for detecting HER2 and RPL30. According to one characteristic of the present invention, the amplification of the transcripts of the HER2 gene and of the control gene is at least qPCR in simplex or multiplexed format thus allowing a better quantification of HER2 and a gain in time and cost.

Selon une autre caractéristique de l'invention, la quantification plus sensible et plus spécifique de HER2 permettra de sélectionner les patientes candidates pour un traitement à l'Herceptin®. Le mot 'HER2' décrit dans la présente invention, représente le gène 'human epidermal growth factor 2' qui code pour la protéine HER2, une thyrosine kinase quij peut induire la métastase et la prolifération tumorale, en cas de surexpression. According to another characteristic of the invention, the more sensitive and specific quantification of HER2 patients will select candidates for treatment with Herceptin ®. The word 'HER2' described in the present invention represents the gene 'human epidermal growth factor 2' which codes for the HER2 protein, a thyrosine kinase which can induce metastasis and tumor proliferation, in case of overexpression.

La détection et la quantification du transcrit du gène HER2 peut supporter le diagnostic et sélectionner les candidates pour le traitement à l'Herceptin®. Detection and quantification of the HER2 gene transcript can support the diagnosis and select candidates for Herceptin ® treatment.

Le mot 'gène' utilisé dans la présente invention réfère à une séquence d'acide nucléique de la molécule d'ADN occupant une région précise dans un chromosome et permet de coder les instructions pour la synthèse de l'ARN. The word 'gene' used in the present invention refers to a nucleic acid sequence of the DNA molecule occupying a specific region in a chromosome and permits the encoding of instructions for RNA synthesis.

Le mot 'oligonucléotide' est une séquence composée d'ADN ou d'ARN ou leur combinaison avec une longueur qui vari de 10 à 70 nucléotides. Les oligonuclétides peuvent être synthétisés par plusieurs méthodes mais pas limitées à celle de la synthèse 5'-3' basée sur l'utilisation des groups protégeant beta-ctanoethyl phosphate (Rosenthal et al, Terahedron Letter 24 : 1691, 1983; Gait et al, Nue Acids Res 4: 1135, 1977) ou la méthode des phosphochloridites (Matteucci J AM CHEM SOC 103: 3185-91, 1981). L'amplification comme utilisé ici réfère à une ou plusieurs méthodes capables de copier un acide nucléique permettant ainsi une augmentation du nombre de copie d'une séquence d'acide nucléique cible. La séquence amplifiée peut être un acide ribonucléotide (ARN) ou un acide desoxyribonucléotide (ADN) Le mot 'transcription inverse' citée ici représente une méthode permettant la synthèse d'ADN complémentaire (ADNc) à partir de molécule d'ARN. L'ADNc sera utilisée dans la méthode PCR, RT-PCR ou qPCR. The word 'oligonucleotide' is a sequence composed of DNA or RNA or their combination with a length that ranges from 10 to 70 nucleotides. Oligonucleotides can be synthesized by several methods but not limited to that of 5'-3 'synthesis based on the use of beta-octanoethyl phosphate protecting groups (Rosenthal et al., Terahedron Letter 24: 1691, 1983; Nude Acids Res 4: 1135, 1977) or the phosphochloridite method (Matteucci J AM CHEM SOC 103: 3185-91, 1981). Amplification as used herein refers to one or more methods capable of copying a nucleic acid thereby allowing an increase in the number of copies of a target nucleic acid sequence. The amplified sequence can be a ribonucleotide acid (RNA) or a deoxyribonucleotide acid (DNA) The word 'reverse transcription' mentioned here represents a method for the synthesis of complementary DNA (cDNA) from RNA molecule. The cDNA will be used in the PCR, RT-PCR or qPCR method.

Le mot 'amorce' réfère à une séquence d'oligonucléotides synthétisée chimiquement ou naturellement. L'amorce est le point d'initiation de la synthèse d'ADN dans des conditions optimales de température et en présence de d'enzyme, tampons, de nucléotides et de séquences nucléotidiques complémentaires spécifiques. ! Le mot 'sonde' citée ici réfère à une séquence nucléotidique qui forme une structure hybride avec une séquence cible dans une molécule d'un échantillon test. Le mot 'PCR' (polymerase chain reaction) comme utilisée dans la présente invention réfère à une méthode dont un échantillon d'ADNc ou d'ADN est ajouté dans une solution en présence de nucléotides non attachés (exemple les dNTPs), 2 oligonucleotides amorces (forward et reverse); et de l'enzyme ADN polymérase (préférablement la Taq polymérase résistant à la chaleur) qui permet la catalyse la formation d'ADN à partir dès 2 amorces et dNTPs. La solution mixe est chauffée à 94-96 C° pour dénaturer la molécule d'ADN et former 2 brins simples d'ADN. Les amorces vont ensuite se fixer spécifiquement sur l'ADN simple brin permettant ainsi à l'ADN polymérase de catalyser l'attachement des dNTPs aux amorces. Le mot 'RT-PCR' (reverse transcriptase-PCR) utilisé dans la présente invention représente une méthode de PCR dont le produit de départ n'est pas directement un ADN mais un ARN traduit en ADNc après transcription inverse. The word "primer" refers to a sequence of oligonucleotides synthesized chemically or naturally. The primer is the point of initiation of DNA synthesis under optimal temperature conditions and in the presence of specific enzyme, buffers, nucleotides and complementary nucleotide sequences. ! The word 'probe' referred to herein refers to a nucleotide sequence that forms a hybrid structure with a target sequence in a molecule of a test sample. The word 'PCR' (polymerase chain reaction) as used in the present invention refers to a method from which a cDNA or DNA sample is added to a solution in the presence of unattached nucleotides (eg dNTPs), 2 oligonucleotide primers (forward and reverse); and the DNA polymerase enzyme (preferably heat-resistant Taq polymerase) that catalyzes the formation of DNA from as early as 2 primers and dNTPs. The mixed solution is heated to 94-96 ° C to denature the DNA molecule and form 2 single strands of DNA. The primers will then bind specifically to the single-stranded DNA thereby allowing the DNA polymerase to catalyze the attachment of the dNTPs to the primers. The word 'RT-PCR' (reverse transcriptase-PCR) used in the present invention represents a PCR method whose starting material is not directly DNA but an RNA translated into cDNA after reverse transcription.

Le mot 'qPCR' (quantitative PCR ou real time RT-PCR) décrit dans la présente invention représente une méthode de PCR permettant l'étude des produits de la réaction PCR pendant les premières étapes d'amplification de l'ADN. La détection des produits de PCR se fait par des sondes marquées à leur extrémité 5' par un fluorochrome émetteur (reporter), et à leur extrémité 3' par un fluorochrome suppresseur (quencher) fluorescent ou non, qui inhibe l'émission du reporter lorsqu'ils sont à proximité. Dans ce cas, l'intensité jdu signal de la fluorescence mesurée au cours la réaction d'amplification est proportionnelle au nombre de produits nouvellement formés (amplicons). La mesure en ADN ou ADNc se fait en logarithme. Pour quantifier un échantillon par qPCR et/ou déterminer la positivité d'une PCR, on détermine le nombre de cycles à partir duquel le produit PCR est détectable. Le moment d'apparition de ce signal seuil dénommé cycle seuil ou Ct (Cycle Threshold) est dépendant de la quantité de matrice initialement présente dans l'échantillon amplifié. Le Ct calculé est inversement proportionnel au logarithme décimal du nombre de copies initiales. Parmi les sondes les plus utilisées en qPCR on trouve les sondes moiecular beacons et TaqMan qui utilisent l'activité exonucléase 5 fluorogénique de l'enzyme Taq polymérase pour mesurer la quantité de la séquence d'ADN dans un échantillon test. Préférentiellement, la qPCR de la présente invention utilise les sondes TaqMan et l'analyse d'amplification est faite par ABI PRISM 7900HT 'séquence détection system' qui est un système de screening pouvant détecter et quantifier des acides nucléiques. La quantité de i The word "qPCR" (quantitative PCR or real time RT-PCR) described in the present invention represents a PCR method for studying the products of the PCR reaction during the first DNA amplification steps. The PCR products are detected by probes labeled at the 5 'end by a transmitter fluorochrome (reporter), and at their 3' end by a fluorescent or non-fluorescent quencher, which inhibits the emission of the reporter when they are nearby. In this case, the signal intensity of the fluorescence measured during the amplification reaction is proportional to the number of newly formed products (amplicons). The measurement in DNA or cDNA is in logarithm. To quantify a sample by qPCR and / or determine the positivity of a PCR, the number of cycles from which the PCR product is detectable is determined. The time of appearance of this threshold signal called threshold cycle or Ct (Cycle Threshold) is dependent on the amount of matrix initially present in the amplified sample. The calculated Ct is inversely proportional to the decimal logarithm of the number of initial copies. Among the most used probes in qPCR are the meecular beacons and TaqMan probes that use the fluorogenic exonuclease activity of the Taq polymerase enzyme to measure the amount of the DNA sequence in a test sample. Preferentially, the qPCR of the present invention uses the TaqMan probes and the amplification analysis is made by ABI PRISM 7900HT 'sequence detection system' which is a screening system that can detect and quantify nucleic acids. The quantity of i

HER2 et du gène contrôle est calculée par le logiciel intégré dans le système ABI PRISM 7900HT en utilisant la méthode de la courbe standard. Selon la présente invention, le reporteur de la sonde peut être FAM, JOE, YAKYE (ΥΎ) et le quencher BBQ, BI-iQl, ou TAMRA.  HER2 and control gene is calculated by the software integrated in the system ABI PRISM 7900HT using the standard curve method. According to the present invention, the probe reporter may be FAM, JOE, YAKYE (ΥΎ) and BBQ quencher, BI-iQl, or TAMRA.

Le mot simplexe de la présente invention représente un essai qui ne se déroule pas simultanément avec d'autres essais. Selon la présente invention, la qPCR en simplexe reflète la détection de nombre de copies d'un seul gène dans un tube de réaction. The simplex word of the present invention represents a test that does not run concurrently with other tests. According to the present invention, simplex qPCR reflects the detection of copy number of a single gene in a reaction tube.

Le mot 'gène contrôle' ou 'housekeeping gene' décrit dans la présente invention représente un gène exprimé en général de façon similaire par toutes les cellules d'un organisme. Parmi les gènes contrôles cités en litérature, on trouve la beta-actine, glyceraldehyd-3-phosphate dehydrogenase (GAPDH), ABL1 et autres. The word "gene control" or "housekeeping gene" described in the present invention represents a gene generally similarly expressed by all cells of an organism. Among the control genes cited in the literature, beta-actin, glyceraldehyd-3-phosphate dehydrogenase (GAPDH), ABL1 and others are found.

Le mot multiplexe cité dans la présente invention réfère à un essai qui se déroule ί The multiplex word cited in the present invention refers to a test that takes place ί

simultanément avec au moins un autre essai. La qPCR en multiplexe préconise une détection du nombre de copies d'au moins 2 gènes dans un même tube de réaction. Selon la présente invention, les 2 gènes HER2 et RPL30 peuvent être détectés simultanément par qPCR. simultaneously with at least one other test. The multiplexed qPCR recommends a detection of the number of copies of at least 2 genes in the same reaction tube. According to the present invention, the 2 genes HER2 and RPL30 can be simultaneously detected by qPCR.

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Le mot 'échantillon test' comme décrit dans la présente invention réfère à un échantillon de biopsie tumoral fraîche ou paraffinée, cellules primaires, lignées cellulaires, salive ou autre fluides organiques exprimant ou surexprimants HER2. En accord avec la présente invention, l'échantillon test peut être d'origine humaine ou animal exprimant ou surexprimant le gène HER2 qui peut être amplifié en utilisant les amorces et sondes de la présente invention. La méthode de qPCR citée dans la présente invention permet de déterminer le nombre de copies du gène HER2 dans un échantillon test en quantifiant le nombre de copies de HER2 relative à un gène contrôle. Le gène contrôle est sélectionné parmi les gènes RLP30, RP37, MPRL19 et tout gène contrôle codant pour des protéines ribosomales. The word "test sample" as described in the present invention refers to a fresh or waxed tumor biopsy sample, primary cells, cell lines, saliva or other organic fluids expressing or HER2 overexpressing. In accordance with the present invention, the test sample can be of human or animal origin expressing or overexpressing the HER2 gene that can be amplified using the primers and probes of the present invention. The qPCR method cited in the present invention makes it possible to determine the number of copies of the HER2 gene in a test sample by quantifying the number of HER2 copies relating to a control gene. The control gene is selected from the genes RLP30, RP37, MPRL19 and any control gene coding for ribosomal proteins.

L'exemple 1 représente une description du protocole utilisé dans la méthode qPCR de la présente invention. Les étapes d'extraction d'ARN, de synthèse d'ADNc et de qPCR sont décrites dans ce protocole. Example 1 represents a description of the protocol used in the qPCR method of the present invention. The steps of RNA extraction, cDNA synthesis and qPCR are described in this protocol.

L'exemple 2: Figure 1 et Figure 2 et Tableau 1 décrivent la haute sensibilité et specifité de la qPCR de la présente invention pour détecter les transcrits du gène HER2 au niveau de la lignée humaine de cancer de sein SKBR3 surexprimant HER2. Example 2: Figure 1 and Figure 2 and Table 1 describe the high sensitivity and specificity of the qPCR of the present invention for detecting HER2 gene transcripts at the HER2-overexpressing SKBR3 breast cancer human line.

Les résultats de la qPCR de la présente invention sont montrés en Figure 1 (courbe d'amplification : cycle-vs-Delta Rn), Figure 2 (courbe standard : quantité d'ADNc de SKBR3- vs-valeurs Ct) et Tableau 1 (valeur de Ct correspondants à la courbe standard). Les valeurs de qualité obtenus sont suivant les normes IVD internationales (R2>0,95, slope entre -3,0 et 3,9 et éfficacité de 90-112)) avec une courbe standard à slope de -3.08, un R2 de 0.98 et une efficacité . de 111 (Figure 2). La courbe d'amplification (Figure 1) montre aussi une allure parfaite.  The results of the qPCR of the present invention are shown in Figure 1 (amplification curve: cycle-vs-Delta Rn), Figure 2 (standard curve: amount of SKBR3 cDNA vs Ct-values) and Table 1 ( value of Ct corresponding to the standard curve). The quality values obtained are according to international IVD standards (R2> 0.95, slope between -3.0 and 3.9 and efficiency of 90-112)) with a standard curve with a slope of -3.08, an R2 of 0.98 and an efficiency. of 111 (Figure 2). The amplification curve (Figure 1) also shows a perfect pace.

L'exemple 3 : Figure 3 montre la courbe d'amplification (cycle-vs-Delta Rn) de la qPCR de la présente invention pour détecter le transcrit du gène HER2 chez des patientes de cancer de sein sur-exprimant HER2 (score 3+) (courbe vert) ou exprimant normalement HER2 (score 0) (courbe bleu) (Figure 3). Les patientes avec le score 3+ montrent bien une faible valeur Ct et donc une quantité plus élevée de HER2 comparée au patientes avec score 0 (Figure 3). L'exemple 4 : Example 3: Figure 3 shows the amplification curve (cycle-vs-Delta Rn) of the qPCR of the present invention for detecting the HER2 gene transcript in HER2-overexpressing breast cancer patients (score 3+ ) (green curve) or expressing normally HER2 (score 0) (blue curve) (Figure 3). Patients with the score 3+ show a low Ct value and thus a higher amount of HER2 compared to patients with score 0 (Figure 3). Example 4:

Figure 4a montre les valeurs de Ct (en triplicata) de la qPCR de la présente invention chez des patientes de cancer de sein avec un score de 3+, 2+ ou 0 (Figure 4). Les valeurs des Ct de la présente qPCR correspondent bien aux valeurs de l'IHC. En effet, pour le score de 3+, les valeurs des Ct sont plus basses (31-33,5). Dans le cas des patientes avec score négatif, les valeurs de Ct sont comprises entre 34 et 38. Alors la lignée SKBR3 sur-exprimant HER2 a une valeur Ct de 27,2 plus proche au 3+. Figure 4b montre les résultats de qPCR sous forme de valeurs 2deltadeltaCt qui est le nombre de foi de surexpression de HER2 par apport à la lignée cellulaire négative pour HER2. Pour la normalisation, le gène contrôle RPL37 a été utilisé. Différent échantillons de biopsies de patiente de cancer de sein ont été testé (samples 1-9 ; n=ll). La lignée cellulaire SKBR3 sur-exprimant HER2 est utilisée ici comme contrôle positif. Toutes les biopsies de patientes testées sur-expriment HER2 par IHC (résultats n'est pas montré ici). Ce dernier résultat a été confirmé dans la présente manip par qPCR et la méthode 2deletadeltaCt (Figure 4b) (2deltadeltaCt des biopsies de patientes > 2deltadeltaCT MCF7). Les enchantions de biopsies 3, 5 et 7 ont une expression de HER2 vingt foi plus supérieur au basale ( CF7). Figure 4a shows the Ct (triplicata) values of the qPCR of the present invention in breast cancer patients with a score of 3+, 2+ or 0 (Figure 4). The Ct values of the present qPCR correspond well to the values of the IHC. Indeed, for the score of 3+, the Ct values are lower (31-33.5). In the case of patients with a negative score, the Ct values are between 34 and 38. Then the over-expressing HER2 SKBR3 line has a Ct value of 27.2 which is closer to 3+. Figure 4b shows the results of qPCR in the form of 2deltadeltaCt values which is the HER2 overexpression index number by contribution to the negative cell line for HER2. For normalization, the RPL37 control gene was used. Different samples of breast cancer patient biopsies were tested (samples 1-9, n = 11). The over-expressing HER2 SKBR3 cell line is used here as a positive control. All biopsies of patients tested overexpress HER2 by IHC (results not shown here). This last result was confirmed in the present manipulation by qPCR and the 2deletadeltaCt method (Figure 4b) (2deltadeltaCt biopsies of patients> 2deltadeltaCT MCF7). Biopsy features 3, 5 and 7 have HER2 expression more than 20 basal (CF7).

L'exemple 5 : Figure 5 montre la courbe des gènes contrôles les plus stables. La mesure de la stabilité de l'expression de plusieurs gènes contrôles par qPCR sur des biopsies de patientes de cancer de sein HER2 a été validée en utilisant le logiciel Genorm. Les gènes contrôles RPL30, RPLP37 et MRPL19 se trouvent tous à droite de la courbe dans la partie des gènes les plus stables (Figure 5). Les mêmes gènes contrôles ont été identifiés aussi comme les plus stables dans les lignées cellulaires de cancer de sein (SKBR3, MCF7, DA) en utilisant le même logiciel Genorm (résultats ne sont pas montrés).  Example 5: Figure 5 shows the curve of the most stable control genes. Measurement of the stability of the expression of several control genes by qPCR on biopsies of HER2 breast cancer patients was validated using the Genorm software. The control genes RPL30, RPLP37 and MRPL19 are all to the right of the curve in the part of the most stable genes (Figure 5). The same control genes were also identified as the most stable in breast cancer cell lines (SKBR3, MCF7, DA) using the same Genorm software (results are not shown).

EXPERIENCES EXPERIENCES

Exemple 1 : Protocole de qPCR de la présente invention pour la quantification de HER2 au niveau de la lignée cellulaire SKBR3 surexprimant HER2 ou des biopsies paraffinées de cancer de sein ! Example 1: qPCR protocol of the present invention for the quantification of HER2 at the HER2 overexpressing SKBR3 cell line or paraffinic breast cancer biopsies !

Le RNeasy mini kit du Qiagen a été utilisé pour extraire l'ARN cellulaire de SKBR3 selon les recommandations du fournisseur (Qiagen Inc). Alors que l'extraction de l'ARN à partir des échantillons de biopsies de patientes de cancer de sein a été faite par le kit Qiagen (QiagenRNAeasy FFPE extraction kit) ou celui d'AMSBIO (England) suivant les recommandations des fournisseurs. La qualité et la pureté de l'ARN obtenue est testée par électrophorèse sur gel d'agarose, ou par le Bioanalyzer (Agilant). The RNeasy mini kit from Qiagen was used to extract SKBR3 cell RNA according to the supplier's recommendations (Qiagen Inc). While RNA extraction from breast cancer patient biopsy samples was done by the Qiagen kit (QiagenRNAeasy FFPE extraction kit) or that of AMSBIO (England) following the recommendations of the suppliers. The quality and purity of the RNA obtained is tested by agarose gel electrophoresis, or by the Bioanalyzer (Agilant).

L'ADN complémentaire (ADNc) est obtenu à partir d'ARN total extrait d'échantillon test. La synthèse d'ADNc est réalisée à l'aide du Thermocycleur (Applied Bios stems) en utilisant le kit RNA to cDNA Reverse Transcription selon les recommandations du fournisseur (Applied Biosystems). The complementary DNA (cDNA) is obtained from total RNA extracted from the test sample. The cDNA synthesis is carried out using the Thermocycler (Applied Bios stems) using the RNA kit to cDNA Reverse Transcription according to the supplier's recommendations (Applied Biosystems).

Les amorces et la sonde des gènes cibles et 5μΙ d'ADNc sont ajoutés au mastermix de la réaction qPCR (TaqMan Fast Universal PCR Master mix : Applied Biosystems) pour un volume final de 25μΙ. The primers and the probe of the target genes and 5 μΙ of cDNA are added to the mastermix of the qPCR reaction (TaqMan Fast Universal Master PCR mix: Applied Biosystems) for a final volume of 25 μΙ.

Dans le cas de la réaction en format simplexe, seulement la sonde et les amorces de HER2 ou gène contrôle sont utilisés dans différent puits de qPCR alors que pour la réaction en format multiplexe, les sondes et les amorces pour HER2 et le gène contrôle sont utilisés simultanément dans le même puits de réaction qPCR.  In the case of the simplex reaction, only the probe and the HER2 or control gene primers are used in different qPCR wells whereas for the multiplexed reaction, the probes and primers for HER2 and the control gene are used. simultaneously in the same qPCR reaction well.

Les conditions du cycle thermal de la qPCR de la présente invention sont divisées en différentes étapes : étape 1 à 95C° pendant 20 secondes; étape 2 (cycle de 50) à 95C° pendant 1 seconde ; et une étape 3 à 60°C pendant 30 secondes. The thermal cycle conditions of the qPCR of the present invention are divided into different steps: step 1 at 95 ° C for 20 seconds; step 2 (cycle 50) at 95 ° C for 1 second; and step 3 at 60 ° C for 30 seconds.

Dans le cas de la courbe standard de la qPCR de la présente invention, des séries de dilutions d'ADNc sont utilisées. Les concentrations utilisées sont 4pg, 20pg, lOOpg, 500pg, 2,5ng, 12,5ng, et 25ng. Pour cette courbe standard, et comme les réglementations international, une valeur R2>0,95 est acceptée par contre une valeur R2<0,95 est refusée et la qPCR doit être répétée. In the case of the standard qPCR curve of the present invention, sets of cDNA dilutions are used. The concentrations used are 4pg, 20pg, 100pg, 500pg, 2.5ng, 12.5ng, and 25ng. For this standard curve, and as international regulations, a value R2> 0.95 is accepted, however, a value R2 <0.95 is refused and the qPCR must be repeated.

Exemples 2, 3, 4a, 4b : La performance de QPCR de la présente invention pour quantifier HER2 dans la lignée de cancer de sein SKBR3 et chez les patients de cancer de sein HER2 (courbe d'amplification, courbe standard, les valeurs de Ct et de 2deltadeltaCt)  Examples 2, 3, 4a, 4b: The performance of QPCR of the present invention for quantifying HER2 in the SKBR3 breast cancer line and in HER2 breast cancer patients (amplification curve, standard curve, Ct values and 2deltadeltaCt)

Les séquences des amorces et de la sonde HER2 utilisées en Figure 1, Figure 2, Figure 3, Figure 4a et Tableau 1 : The sequences of primers and HER2 probe used in Figure 1, Figure 2, Figure 3, Figure 4a and Table 1:

- Amorce Forward : 5' Aatgccaggcactgtttg (SEQ ID N° 1)  - Forward Primer: 5 'Aatgccaggcactgtttg (SEQ ID NO: 1)

- Amorce reverse : 5' Gtccttatagtgggcacagg 3' (SEQ ID N° 2)  Reverse primer: 5 'Gtccttatagtgggcacagg 3' (SEQ ID No. 2)

- Sonde : 5' R- Gtccttatagtgggcacagg-Q 3' (SEQ ID INT. 9)  - Probe: 5 'R-Gtccttatagtgggcacagg-Q 3' (SEQ ID INT.9)

Pour la sonde : R= YY et Q=BHQ1 For the probe: R = YY and Q = BHQ1

i  i

I SEQUENCE LISTING i I SEQUENCE LISTING i

<110> MOROCCAN FOUNDATION FOR ADVANCED SCIENCE INNOVATION & <110> MOROCCAN FOUNDATION FOR ADVANCED SCIENCE INNOVATION &

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<120> Sondes et amorces pour détecter le gène HER2 et un gène (ribosomale) de contrôle en format multiplex : Applications dans le choix de traitement du cancer de sein HER2 <130> PCT/MA2013/000023 <120> Probes and primers for detecting the HER2 gene and a control (ribosomal) gene in multiplex format: Applications in the choice of breast cancer treatment HER2 <130> PCT / MA2013 / 000023

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Claims

Méthode pour la détection et la quantification du gène ou du transcrit du gène HER2 et du gène contrôle spécifique dans un échantillon tes caractérisée en ce que la méthode comprend les étapes suivantes : Method for the detection and quantification of the gene or transcript of the HER2 gene and the specific control gene in a sample, characterized in that the method comprises the following steps: - Extraction de l'ARN à partie d'un échantillon test de la biopsie du patient - Extraction of the RNA from a test sample of the patient's biopsy - Test de pureté et de la quantité de l'ARN par électrophorèse, nanodrop ou par bioanalyseur - Test of purity and quantity of RNA by electrophoresis, nanodrop or bioanalyzer - Synthèse de l'ADN complémentaire (ADNc) à partir de l'ARN total extrait de l'échantillon test et réalisé à l'aide du thermocycleur. .  Synthesis of the complementary DNA (cDNA) from the total RNA extracted from the test sample and carried out using the thermocycler. . Ajout de 5 μΙ d'ADNc, d'une amorce «forward» ayant jdes séquences sélectionnées parmi le group de séquences : [SEQ ID N° 1 ,SEQ ID N° 3, SEQ ID N° 5, SEQ ID N° 7], d'une amorce «reverse» sélectionnée parmi le group de séquences [SEQ ID N° 2, SEQ ID N° 4, SEQ ID N° 6, SEQ ID N°8] et d'une sonde de détection sélectionnée parmi le group de séquences [SEQ ID N° 9, SEQ ID N° 10, et SEQ ID N° 11] au master mix de la réaction qPCR pour un volume final d'au moins 25μΙ.  Addition of 5 μl of cDNA, a "forward" primer with sequences selected from the group of sequences: [SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7] , a "reverse" primer selected from the group of sequences [SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8] and a detection probe selected from the group sequence [SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11] to the master mix of the qPCR reaction for a final volume of at least 25μΙ. - Application du cycle thermal de la qPCR  - Application of the qPCR thermal cycle Mesure de l'amplification du gène  Measurement of gene amplification Méthode selon la revendication 1, où deux amorces pour amplifier le transcrit du gène contrôle RPL30 dans l'échantillon test sont constituées d'une amorce «forward»
Figure imgf000018_0001
The method according to claim 1, wherein two primers for amplifying the transcript of the RPL30 control gene in the test sample consist of a "forward" primer.
Figure imgf000018_0001
 Méthode selon la revendication 1, où une sonde pour détecter le transcrit du gène i  The method of claim 1, wherein a probe for detecting the gene i transcript contrôle RPL30 dans un échantillon test, a une séquence sélectionnée parmi les SEQ ID N"10, SEQ ID °11. RPL30 control in a test sample, has a sequence selected from SEQ ID No. 10, SEQ ID No. 11. Méthode selon la revendication 1, où le gène contrôle (housekeeping gene) peut être sélectionné parmi le groupe des gènes RPLP37, MRPL19 ou tout autre gène contrôle codant pour des protéines de la sous unité ribosomale 60S. The method of claim 1, wherein the housekeeping gene can be selected from the group of RPLP37, MRPL19 genes or any other control gene encoding 60S ribosomal subunit proteins. 5. Méthode selon les revendications 1 à 6, où Les sondes pour détecter le transcrit des gènes HER2 ou RPL30 en qPCR sont attachées à des entités fluorescentes (reporters) et des inhibiteurs de fluorescence (quenchers). Le reporteur peut être FAM, JOE ou YY ou similaires, attaché à l'extrémité 5' et l'inhibiteur de fluorescence (quencher) peut être BBQ, BHOJL, ou TAMRA ou similaires attaché à l'extrémité 3'. The method according to claims 1 to 6, wherein the probes for detecting the transcript of the HER2 or RPL30 genes in qPCR are attached to fluorescent entities (reporters) and fluorescence inhibitors (quenchers). The reporter may be FAM, JOE or YY or the like, attached to the 5 'end and the fluorescence inhibitor (quencher) may be BBQ, BHOJL, or TAMRA or the like attached to the 3' end. 6. Méthode selon les revendications 1 à 7, où la détection des transcrits des gènes HER2 et du gène contrôle d'un échantillon test est effectuée de façon séparée (format simplexe) ou simultanée dans un même puits de qPCR (format multiplex).  6. Method according to claims 1 to 7, wherein the detection of transcripts of HER2 genes and the control gene of a test sample is performed separately (simplex format) or simultaneously in the same well of qPCR (multiplex format). 7. Méthode selon les revendications 1 à 8, où le degré d'expression est mesuré à l'échelle d'ARNm  7. Method according to claims 1 to 8, wherein the degree of expression is measured at the mRNA scale 8. Méthode selon les revendications 1 à 9, où l'utilisation de ladite méthode comprend au moins une des techniques PCR, PCR en temps réel (qPCR) ou transcription inverse PCR (RT-PCR).  The method according to claims 1 to 9, wherein the use of said method comprises at least one of PCR, real-time PCR (qPCR) or reverse transcription PCR (RT-PCR) techniques.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016060536A1 (en) * 2014-10-17 2016-04-21 Morrocan Foundation For Advanced Science Innovation & Research (Mascir) Breast cancer diagnostic kit using multiplex qpcr reaction and use thereof for assessing the status of the her2 protein and for selecting a treatment
WO2019132640A1 (en) * 2017-12-29 2019-07-04 Moroccan Foundation For Advanced Science, Innovation And Research (Mascir) Method for determining the positivity threshold of the overexpression of the her2 protein in breast cancer and other cancers.

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