WO2014129975A1 - Identification de signatures de microarn en circulation pour la détection du cancer du sein - Google Patents

Identification de signatures de microarn en circulation pour la détection du cancer du sein Download PDF

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WO2014129975A1
WO2014129975A1 PCT/SG2014/000073 SG2014000073W WO2014129975A1 WO 2014129975 A1 WO2014129975 A1 WO 2014129975A1 SG 2014000073 W SG2014000073 W SG 2014000073W WO 2014129975 A1 WO2014129975 A1 WO 2014129975A1
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mir
micrornas
microrna
breast cancer
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Kok Leong Maurice CHAN
Siew Gek Ann LEE-LIM
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Singapore Health Services Pte Ltd
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
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    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to methods for screening subjects for breast cancer. More particularly, the present invention relates to screening subjects for the presence of particular microRNA's which have diagnostic efficacy.
  • Breast cancer remains the leading cause of mortality in women, despite improvements in cancer screening and treatment strategies.
  • Mammography is the current gold standard for breast cancer detection, but can have false negative rates of up to 20% (NCI data).
  • NCI data The diagnosis of breast cancer relies on the histological examination of tissue biopsies, or cytology of fine-needle aspirates, which are both invasive procedures.
  • Known serum-based tumour markers such as CA15.3 or BR27.29, cannot be used for breast cancer detection due to their low sensitivity (1 ). There is thus a need to develop novel markers that are minimally invasive, for the improved detection, diagnosis, and molecular understanding of breast cancer.
  • MicroRNAs are approximately 22 nt long non-coding RNAs that can base pair specifically with target mRNAs to induce gene silencing through specific mechanisms involving translational repression or transcript degradation. Since their discovery in 1993, microRNAs have been estimated to regulate more than 60% of all human genes, with many microRNAs identified as key players in critical cellular functions such as proliferation and apoptosis. The current database of microRNAs, MirBase release 19, has >2000 entries of human microRNAs, constituting a major class of regulatory molecules. lorio et al. provided the earliest observation that microRNAs are differentially expressed in breast cancer tumors as compared to normal breast tissue (2).
  • MicroRNAs Analysis of 76 breast cancer tumours and 10 normal samples (non-cancerous breast tissues) using microarrays which probed for 386 microRNAs, identified 29 dysregulated microRNAs.
  • miR-21 and miR-155 were up regulated while miR-10b, miR-125b, and miR-145 were down regulated.
  • Persson and co-workers (3) performed extensive next-generation microRNA sequencing of paired tumour and normal tissue from 5 breast cancer patients, and detected more than 500 microRNAs, including a novel microRNA (miR-4728) encoded within the human epidermal growth factor receptor 2 (Her2) gene, which was overexpressed in Her2 amplified tumours.
  • microRNAs that were differentially expressed depending on breast cancer subtype, histological grade, cancer aggressiveness (4), metastasis-free survival (5), as well as estrogen receptor (ER) (6, 7), Her2 (6, 7), or triple-negative status (4, 6).
  • ER estrogen receptor
  • Circulating microRNAs have been suggested to be able to distinguish breast cancer samples from healthy controls. These studies have usually involved targeted analyses of only 4 to 6 microRNAs by RT-PCR (8, 9). However, comparisons between these studies may not be straightforward as they were carried out under diverse experimental conditions. For example, circulatory microRNAs may have been extracted from serum (8, 9), plasma (11 ), circulating tumour cells, or even whole blood (12, 13). Further, while most studies employed serum samples collected pre-operatively as it has been suggested that microRNA levels may return to baseline within 2 weeks after tumour resection, one other study utilized postoperative sera (8).
  • Circulating microRNAs may also exhibit racial differences, as the microarray profiling of microRNAs in the plasma of 10 cases each from Caucasian and African breast cancer patients resulted in only 2 common dysregulated microRNAs between these groups (10). In contrast to targeted studies involving specific microRNAs, there are few comprehensive profiling studies of circulatory microRNAs in breast cancer (10, 14), and a consistent diagnostic signature for circulatory microRNAs is not yet available. Few studies have attempted to compare the circulatory microRNA profile to that within the breast cancer tumour, such that the relationship between these two profiles of microRNAs is not clear. One study assessed a panel of seven microRNAs while another analyzed five microRNAs.
  • a recent study (16) investigated the status of four plasma-derived microRNAs in matched tumours, and concluded that microRNAs generally displayed opposite expression patterns in tissue and plasma. However, these comparisons between circulating and tumour microRNA profiles were not comprehensive, as microRNA profiling of the serum or plasma samples were not done.
  • novel microRNA expression signatures identified in this study had sufficient diagnostic efficacy for development into blood-based biomarkers for breast cancer detection.
  • the present invention provides a method of detecting whether a subject has breast cancer, comprising screening a blood sample from the subject for the presence of at least one microRNA differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group
  • microRNAs in Table 3 comprising the microRNAs in Table 3, and wherein detection of one or more of said microRNAs indicates breast cancer in the subject.
  • the at least one differentially expressed microRNA is selected from the group comprising the microRNAs in Table 2.
  • the at least one differentially expressed microRNA is selected from the group comprising the microRNAs miR-1 , miR-92a, miR-133a and miR-133b.
  • the subject's blood sample is screened for the differential expression of at least two microRNAs selected from the group comprising: a) miR-92a and miR-1 , b) miR-92a and miR-133a, and c) miR-92a and miR-133b.
  • the microRNA detection method involves using a screening platform selected from, for example, the group comprising cDNA microarrays, oligonucleotide microarrays, microRNA
  • RNA arrays arrays, high throughput quantitative polymerase chain reaction (qPCR), and solution hybridization/ribonuclease digestion kit and probes.
  • Another aspect of the invention provides a method of detecting whether a subject has breast cancer with clinicopathological features of one or more of estrogen receptor (ER) positivity, human epidermal growth factor receptor 2 (Her2) positivity and Node positivity, comprising screening a blood sample from the subject for the presence of at least one microRNA differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • ER estrogen receptor
  • Her2 human epidermal growth factor receptor 2
  • the at least one microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • the subject has already been, or is to be, screened for breast cancer by the method of the invention described supra.
  • kits for screening a blood sample for the presence of microRNA indicative of breast cancer wherein the microRNA is differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, comprising oligonucleotide primers and/or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one differentially expressed microRNA selected from the group comprising the microRNAs in Table 3.
  • the at least one differentially expressed microRNAs are selected from the group comprising the microRNAs in Table 2.
  • the at least one differentially expressed microRNAs are selected from the group comprising the microRNAs miR- 1 , miR-92a, miR-133a and miR-133b.
  • the kit comprises primers and/or probes to detect at least two microRNAs selected from the group comprising: a) miR-92a and miR-1 , b) miR-92a and miR-133a, and c) miR-92a and miR-133b.
  • the kit comprises a screening platform selected from, for example, the group comprising cDNA microarrays, oligonucleotide microarrays, microRNA (miRNA) arrays, high
  • the kit comprises a MicroRNA array, a LNA RT- PCR panel, or a solution hybridization kit specifically directed to the differentially expressed microRNAs of the invention.
  • Another aspect of the invention provides a kit for screening a blood sample for the presence of microRNA indicative of breast cancer which is clinicopathologically ER positive, Her2 positive and/or Node positive, wherein the microRNA is
  • differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease comprising oligonucleotide primers and or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one microRNA selected from the the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • said at least one microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • the kit comprises a screening platform selected from, for example, the group comprising cDNA
  • microarrays oligonucleotide microarrays, microRNA (miRNA) arrays, high
  • the kit comprises a MicroRNA array, a LNA RT- PCR panel, or a solution hybridization kit specifically directed to the differentially expressed microRNAs of the invention.
  • the kit of the invention may further comprise a nucleotide polymerizing enzyme and a reagent buffer.
  • the blood sample is from a subject that has already been, or is to be, screened for breast cancer by the methods described supra.
  • the blood sample is a serum sample.
  • Another aspect of the invention provides a microarray for detecting breast cancer in a subject, comprising a substrate and a set of genetic marker molecules attached to the substrate, wherein the marker molecules are oligonucleotide probes which can identify at least one of the differentially expressed microRNAs of the invention defined in Table 3.
  • the at least one microRNAs are those defined in Table 2.
  • the at least one microRNAs are selected from the group comprising miR-1 , miR-92a, miR-133a and miR-133b.
  • the microarray comprises primers or probes to detect at least two microRNAs selected from the group comprising: a) miR-92a and miR-1, b) miR-92a and miR-133a, and c) miR-92a and miR-133b.
  • Another aspect of the invention provides a microarray for detecting breast cancer in a subject which is clinicopathologically ER positive, Her2 positive and/or Node positive, comprising a substrate and a set of genetic marker molecules attached to the substrate, wherein the marker molecules are oligonucleotide probes which can identify at least one differentially expressed microRNA selected from the the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • said at least one differentially expressed microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • the subject's blood sample is a serum sample.
  • Another aspect of the invention provides a method of detecting whether a subject has breast cancer, comprising screening a suspected breast cancer tissue sample from the subject for the presence of at least one microRNA differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR- 376c and miR-4324, and wherein detection of increased levels of one or more of miR-720, miR-1274b and miR-1260 or detection of decreased levels of one or more of miR-30c, miR-376c and miR-4324 indicates breast cancer in the subject.
  • Another embodiment of the invention provides a method of detecting whether a subject has breast cancer, comprising screening a suspected diseased breast tissue sample from the subject for the presence of at least one microRNA
  • the at least one microRNA is selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR-376c and miR-4324, and wherein detection of increased levels of one or more of miR-720, miR-1274b and miR-1260 or detection of decreased levels of one or more of miR-30c, miR-376c and miR-4324 in the suspected diseased tissue relative to the adjacent tissue indicates breast cancer in the subject.
  • kits for screening a breast tissue sample for the presence of microRNA indicative of breast cancer wherein the microRNA is differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, comprising oligonucleotide primers or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one differentially expressed microRNA selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR-376c and miR-4324.
  • the subject is of Chinese ancestry.
  • Figure 1 Three components principal component analysis of (a) tissue and (b) serum samples.
  • Breast cancer tumour tissues (a) and serum from breast cancer patients (b) are indicated in red T,).
  • Adjacent normal tissues (a) and serum from healthy individuals (b) are indicated in blue (B).
  • Figure 2 Hierarchical clustering of (a) tissues and (b) serum samples, (a) breast cancer tumour and adjacent normal tissues are indicated with red (®) and blue ( ⁇ ) circles, respectively; (b) Serum from breast cancer patients and healthy individuals are indicated with red (it) and blue ( ⁇ ) circles, respectively.
  • Figure 3 Correlation plots from inter-platform comparison studies, (a) same breast cancer tumour sample extracted by mirVana and miRNeasy; (b) same breast cancer tumour sample ran on microarray and RT-PCR panels.
  • Figure 4 Test for collinearity by Variance Inflation Factor (VIF) computation, and derivation of diagnostic models and significant markers by logistic regression (LR). Odds ratio (OR), which is also the exponentiation of the B coefficient. Statistical significance is represented by the P-value.
  • VIP Variance Inflation Factor
  • LR logistic regression
  • OR Odds ratio
  • Figure 5 Validation of significant microRNAs by RT-PCR using 132 cases and 101 controls, (a) - (d) show box-and-whisker plots (generated by PASW) representing RT-PCR results for miR-1 , miR-92a, miR- 33a, and miR-133b respectively.
  • the y-axis depicts log2 fold change.
  • the lines inside the boxes denote the medians.
  • the boxes mark the interval between the 25 th and 75 th percentiles.
  • the whiskers denote the interval between the maximum and minimum values. Filled circles indicate outliers, defined as values beyond one and a half box lengths from either end of the box.
  • Statistical significance was determined using the Mann- Whitney test; (e) ROC curves plotted using the microRNA combinations derived by logistic regression.
  • the term 'differentially expressed' refers to increased (up regulated) or decreased (down regulated) expression of microRNA in a diseased subject relative to the expression level in the respective normal disease-free state.
  • the microRNA expression levels in the serum of a test subject may be compared to that in the serum of a subject absent the disease or cohort of subjects absent the disease.
  • the microRNA expression levels in a suspected cancer tissue of a test subject may be compared to that in an adjacent 'disease-free' tissue sample of the test subject or to a corresponding tissue of a different subject absent the disease or cohort of subjects absent the disease.
  • the term 'a subject absent the disease' refers to a subject who is considered to be free of breast cancer for the purpose of the invention.
  • the present invention provides a method of detecting whether a subject has breast cancer, comprising screening a blood sample from the subject for the presence of at least one microRNA differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group comprising the microRNAs in Table 3, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer in the subject.
  • An advantage of using a blood sample from the subject is that it is not a very invasive procedure on the body compared to, for example, the removal of tissue specimens by biopsy for analysis. Moreover, blood samples are often taken for other tests so the inconvenience to the subject can be minimised.
  • the at least one differentially expressed microRNA is selected from the group comprising the microRNAs in Table 2.
  • the at least one differentially expressed microRNA is selected from the group comprising the microRNAs miR-1 , miR-92a, miR-133a and miR-133b.
  • the nucleotide sequences of miR- 1 , miR-92a, miR-133a and miR-133b are represented by SEQ ID NOS: 1 , 2, 3 and 4, respectively.
  • AUCs areas under the curves
  • the subject's blood sample is screened for the differential expression of at least two microRNAs selected from the group comprising: a) miR-92a and miR-1 , b) miR-92a and miR-133a, and c) miR-92a and miR-133b.
  • the microRNA detection method involves using a screening platform selected from, for example, the group comprising cDNA microarrays, oligonucleotide microarrays, microRNA
  • MicroRNA arrays and LNA RT-PCR panels such as those described in the Examples herein may be suitable for the methods of the invention.
  • Solution hybridization kits such as the m/ ' rVanaTM miRNA Detection Kit manufactured by Life Technologies Corporation may also represent a suitable screening platform. It is to be understood by a person skilled in the art that the screening platform used in the methods of the invention is not to be limited to a selection from those described above.
  • Another aspect of the invention provides a method of detecting whether a subject has breast cancer with clinicopathological features of one or more of estrogen receptor (ER) positivity, human epidermal growth factor receptor 2 (Her2) positivity and Node positivity comprising screening a blood sample from the subject for the presence of at least one microRNA differentially expressed in a diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • ER estrogen receptor
  • Her2 human epidermal growth factor receptor 2
  • said at least one microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • nucleotide sequences of the microRNAs listed in Table 4 are represented in Table 8 by SEQ ID NOS: 5 to 31.
  • the subject has already been, or is to be, screened for breast cancer by the method described supra.
  • kits for screening a blood sample for the presence of microRNA indicative of breast cancer wherein the microRNA is differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, comprising oligonucleotide primers or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one differentially expressed microRNA selected from the group comprising the microRNAs in Table 3.
  • the at least one differentially expressed microRNAs are selected from the group comprising the microRNAs in Table 2.
  • the at least one differentially expressed microRNAs are selected from the group comprising the microRNAs miR- 1 , miR-92a, miR-133a and miR-133b.
  • the nucleotide sequences of miR-1 , miR-92a, miR-133a and miR-133b are represented by SEQ ID NOS: 1 , 2, 3 and 4, respectively.
  • AUCs areas under the curves
  • the kit comprises primers or probes to detect at least two microRNAs selected from the group comprising: a) miR-92a and miR-1 , b) miR-92a and miR-133a, and c) m ' iR-92a and m ' iR-133b.
  • the kit comprises a screening platform selected from, for example, the group comprising cDNA microarrays, oligonucleotide microarrays, microRNA (miRNA) arrays, high
  • the kit comprises a MicroRNA array, a LNA RT- PCR panel, qPCR primers, or a solution hybridization kit specifically directed to the differentially expressed microRNAs of the invention.
  • the kit may also comprise software/protocol for data interpretation.
  • Another aspect of the invention provides a kit for screening a blood sample for the presence of microRNA indicative of breast cancer which is clinicopathologically ER positive, Her2 positive and/or Node positive, wherein the microRNA is
  • differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease comprising oligonucleotide primers and or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one microRNA selected from the the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein detection of differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • said at least one microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • the nucleotide sequences of the microRNAs listed in Table 4 are represented in Table 8 by SEQ ID NOS: 5 to 31.
  • the kit comprises a screening platform selected from, for example, the group comprising cDNA microarrays, oligonucleotide microarrays, microRNA (miRNA) arrays, high throughput quantitative polymerase chain reaction (qPCR), and solution
  • the kit comprises a MicroRNA array, a LNA RT- PCR panel, qPCR primers, or a solution hybridization kit specifically directed to the differentially expressed microRNAs of the invention.
  • the kit of the invention may further comprise a nucleotide polymerizing enzyme and a reagent buffer.
  • the blood sample is from a subject that has already been, or is to be, screened for breast cancer by the methods described supra.
  • Another aspect of the invention provides a microarray for detecting breast cancer in a subject, comprising a substrate and a set of genetic marker molecules attached to the substrate, wherein the marker molecules are oligonucleotide probes which can identify at least one of the differentially expressed microRNAs of the invention defined in Table 3.
  • the at least one microRNAs are those defined in Table 2.
  • the at least one microRNAs are selected from the group comprising miR-1 , miR-92a, miR-133a and miR-133b.
  • the nucleotide sequences of miR-1 , miR-92a, miR-133a and miR- 133b are represented by SEQ ID NOS: 1 , 2, 3 and 4, respectively.
  • the microarray comprises primers or probes to detect at least two microRNAs selected from the group comprising: a) miR-92a and miR-1, b) miR-92a and miR-133a, and c) miR-92a and miR-133b.
  • Another aspect of the invention provides a microarray for detecting breast cancer in a subject which is clinicopathologically ER positive, Her2 positive and/or Node positive, comprising a substrate and a set of genetic marker molecules attached to the substrate, wherein the marker molecules are oligonucleotide probes which can identify at least one differentially expressed microRNA selected from the the group comprising: a) microRNAs in Table 6 for ER, b) microRNAs in Table 6 for Her2, and c) microRNAs in Table 6 for node positivity, and wherein differential expression of one or more of said microRNAs indicates breast cancer with said one or more clinicopathological features in the subject.
  • said at least one differentially expressed microRNA is selected from the group comprising: a) microRNAs in Table 4 for ER, b) microRNAs in Table 4 for Her2, and c) microRNAs in Table 4 for node positivity.
  • nucleotide sequences of the microRNAs listed in Table 4 are represented in Table 8 by SEQ ID NOS: 5 to 31.
  • the subject's blood sample is a serum sample.
  • Another aspect of the invention provides a method of detecting whether a subject has breast cancer, comprising screening a suspected breast cancer tissue sample from the subject for the presence of at least one microRNA differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, wherein the at least one microRNA is selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR- 376c and miR-4324, and wherein detection of increased levels of one or more of miR-720, miR-1274b and miR-1260 or detection of decreased levels of one or more of miR-30c, miR-376c and miR-4324 indicates breast cancer in the subject.
  • Another embodiment of the invention provides a method of detecting whether a subject has breast cancer, comprising screening a suspected diseased breast tissue sample from the subject for the presence of at least one microRNA
  • the at least one microRNA is selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR-376c and miR-4324, and wherein detection of increased levels of one or more of miR-720, miR-1274b and miR-1260 or detection of decreased levels of one or more of miR-30c, miR-376c and miR-4324 in the suspected diseased tissue relative to the adjacent tissue indicates breast cancer in the subject.
  • kits for screening a tissue sample for the presence of microRNA indicative of breast cancer wherein the microRNA is differentially expressed in the diseased state compared to a reference sample representing a subject absent the disease, comprising oligonucleotide primers or probes capable of binding to and/or amplifying at least a portion of the nucleic acid sequence, or cDNA derived therefrom, of at least one differentially expressed microRNA selected from the group comprising the microRNAs miR-720, miR-1274b, miR-1260, miR-30c, miR-376c and miR-4324.
  • nucleotide sequences of these miRNAs are represented in Table 8 by SEQ ID NOS: 32-37.
  • the subject is of Chinese ancestry.
  • Matched fresh frozen breast cancer tumours, adjacent normal tissues, and pre-operative sera from 32 breast cancer patients were obtained from the SingHealth Tissue Repository. Control serum samples were recruited from 22 healthy female volunteers. The mean age ⁇ standard deviation for the patients at diagnosis, and healthy volunteers at time of recruitment, were 50 ⁇ 13 years and 47 ⁇ 6 years, respectively. Of the 32 breast cancer patients employed for the profiling stage, 3 (9%), 15 (46%), 9 (28%), or 2 (6%), were diagnosed with stage 1 , 2, 3, or 4 cancer, respectively.
  • ILC Invasive lobular carcinoma
  • LN lymph node status
  • MicroRNAs were extracted from tissue or serum samples using the miRVanaTM (Life Technologies, Carlsbad, CA) or miRNeasy (Qiagen, Hilden, Germany) kits respectively, according to manufacturers' instructions.
  • miRNeasy the standard protocol was modified based on Exiqon's application note "RNA Purification from Blood Plasma & Serum" (located on their web site), which used MS2 (Roche, Basel, Switzerland) as a carrier.
  • MicroRNA extraction was carried out using 6 to 10 pieces of tissue (approximately 1x1x1 mm) or 250ul of serum as the starting material.
  • Quality control (QC) of RNA from tissue samples was carried out using the Agilent Bioanalyzer (Santa Clara, CA).
  • QC of serum samples was carried out using single-plex LNATM RT-PCR (Exiqon, Vedbaek, Denmark), and LNA primers for serum markers (miR-16 and miR-20a).
  • reverse transcription was carried out using the Universal cDNA Synthesis kit (Exiqon), employing 4ul of microRNA-containing total RNA, 2ul of enzyme mix, and 4ul of 5x reaction buffer, made up to a 20ul reaction volume using nuclease-free water. Reverse transcription was carried out at 42°C for 60 min, followed by inactivation at 95°C for 5 mins. Every RT-PCR experiment included no reverse transcription controls.
  • 10ul reactions were prepared in the following proportions: 5ul of SYBR Green master mix, 1ul of LNA primer mix, and 4ul of cDNA template (55x dilution).
  • RT-PCR was performed at 95°C for 10min; followed by 40 cycles of 95°C for 10s/60°C for 1 min using an Applied BiosystemsTM 7500 Real-Time PCR System (Life Technologies). MicroRNA microarray and LNA RT-PCR panels
  • the Agilent human microRNA microarray was based on miRBase Release 16.0, with probes for about 1300 microRNAs.
  • the microarray is based on a direct labeling (Cy3) chemistry, and was carried out according to the manufacturer's standard protocol. Each microarray experiment employed 200ng of microRNA-containing total RNA.
  • the LNATM RT-PCR human microRNA panels comprised of two 384- well plates for the detection of 742 microRNAs. Reverse transcription was performed using the Universal cDNA Synthesis kit (Exiqon) in 40ul reactions per panel, employing 8ul of microRNA-containing total RNA, 4ul of enzyme mix, and 8ul of 5x reaction buffer, made up to 40ul using nuclease-free water. For each 384-well plate, the cDNA was diluted 55x (using 2160ul of nuclease-free water). Two ml of the diluted cDNA was combined with an equal volume of 2x SYBR Green Master Mix (Exiqon) and dispensed at 10ul per well. The RT-PCR was executed according to Exiqon's protocol for serum and plasma on an Applied BiosystemsTM 7900HT Real- Time PCR System (Life Technologies) which was set using run templates (SDS files) downloaded from Exiqon's website.
  • SDS files run templates
  • the GEO accession number for the microRNA expression profiles from the microarray and RT-PCR panels reported in this study is GSE42128.
  • Microarray expression data was imported into the GeneSpring software (Agilent). Global normalization was carried out based on 90 percentile shift followed by log2 transformation. Principal component analysis (PCA), paired and unpaired t- test, and cluster analysis were computed using the GeneSpring software.
  • PCA Principal component analysis
  • Ct values from RT-PCR were imported into the GenEx software (Exiqon).
  • the analysis workflow included (i) QC using no reverse transcription controls, (ii) interplate calibration, (iii) selection of reference genes using NormFinder and GeNorm, and (iv) normalization and log2 transformation.
  • PCA, cluster analysis, f-test (unpaired, 2-tailed), Mann-Whitney test (2-sided), and Kolmogorov-Smirnov test (for normal distribution) were done using the GenEx software where appropriate.
  • VIF Variance Inflation Factor
  • MicroRNA profiling of tumor and adjacent normal tissue samples Significant differentially expressed microRNAs were identified by applying the paired i-test (23 pairs of breast cancer tumours vs. adjacent normal tissues) or the unpaired i-test (31 breast cancer tumours vs. 23 adjacent normal tissues). This resulted in 73 microRNAs that were significant (p ⁇ 0.05) after correction for multiple testing by Benjamini-Hochberg FDR (false discovery rate) in both paired as well as unpaired f-tests. The 20 most significant microRNAs, with corrected P values ranging from 1.6E-06 to 8.0E-09, are shown in Table 2.
  • Table 4 lists the microRNAs that were significantly associated with ER, Her2, and lymph node positivity, as determined using the unpaired Student's f-test, without correction for FDR. Interestingly, almost all of the differentially expressed microRNAs were novel insofar as being associated with breast cancer, with the majority being unique from those identified in other studies (4-7). Notably, these previous studies did not share common significant microRNAs between each other.
  • both the geNorm and NormFinder algorithms identified miR-103 and miR-191 as the most stably expressed, best gene combination for use as reference genes for normalizing the RT-PCR data.
  • Statistical analysis of the serum microRNA profiles led to the identification of 85 microRNAs that were significant (p ⁇ 0.05) after FDR correction for multiple testing. The most significant 20 microRNAs are shown in Table 2, and 18 of these were up regulated in breast cancer. Most of these microRNAs appeared to be novel and have not been reported in the context of circulating microRNA in breast cancer. A complete list of significant microRNAs identified from serum is provided in Table 3. TABLE 4. Ten most significant breast cancer tumour microRNAs and serum microRNAs associated with clinicopathological features.
  • miR-203 0.018 down 1.60 miR-484 0.019 up 1.81
  • miR-10a 0.019 up 1.17 miR-1306 0.023 down 2.08
  • miR-652 0.020 up 0.78 miR-129 * 0.025 up 1.50
  • miR-342-5p 0.020 up 1.14 miR-374c 0.028 up 1.83
  • miR-4284 0.020 down 0.96 miR-629* 0.030 up 3.63
  • miR-29b-1* 0.020 down 0.62 miR-16-2* 0.035 up 1.55
  • serum microRNAs differentially expressed according to ER, Her2 and lymph node status could also be identified (Table 4), using the unpaired f-test without correction for FDR.
  • Table 4 A complete list of serum microRNAs differentially expressed according to ER, Her2 and lymph node status is shown in Table 6, with their nucleotide sequences, obtained from the miRBase (microRNA data base) website, in Table 8.
  • nucleotide sequences of these miRNAs can be found, for example, in the miRBase website and are represented in Table 8. TABLE 8. Nucleotide sequence listing of microRNAs
  • microRNAs that were up regulated miR-720, miR- 274b and miR- 260
  • down regulated miR-30c, miR-376c and miR-4324
  • the nucleotide sequences of these miRNAs can be found, for example, in the miRBase website and are represented in Table 8 by SEQ ID NOS: 32-37.
  • this study represents the largest serum and tumour cohort in terms of extensive profiling of microRNAs.
  • two other studies (11 , 16) profiled 20 samples in their marker discovery stage.
  • the use of appropriate normalization controls is a well-known crucial issue for RT-PCR experiments.
  • the use of a larger profiling cohort in this study facilitated the selection of reference microRNAs empirically.
  • the use of a spike-in or a small RNA for data normalization in similar studies ( 1, 16) have sometimes been considered to be problematic due to their suspected instability (21).
  • Intracellular ⁇ , miR-1 , miR-92a, miR- 33a and miR- 33b appear to play tumour suppressor roles in cancer cells. It is not known whether these microRNAs have anti-tumorigenic properties in their circulating forms. The presence of circulating microRNAs has only been recognized over the last few years, and the understanding of their biological roles is just emerging. Circulating microRNAs have been proposed to play either oncogenic or tumour suppressive roles (21). For example, exosomes containing microRNAs derived from human melanomas and colorectal carcinomas were able to promote tumour growth and immune escape. Alternatively, immunocytes may secrete tumour suppressive microRNAs so as to block tumor proliferation or promote apoptosis (21 ).
  • the 20 most significant microRNAs differentially expressed in breast cancer tumours included miR-21 , miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Only seven microRNAs were overexpressed in both tumours and serum, suggesting that microRNAs may be released into the serum 4g selectively. Interestingly, 16 of the 20 most significant microRNAs differentially expressed in serum samples were novel.
  • MiR-1 , m " iR-92a, miR-133a and miR- 33b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these microRNAs exhibited areas under the curves of 0.90-0.91.
  • the term 'comprising does not preclude the presence of additional steps or substances in the methods and compositions, respectively, of the invention, and is understood to include within its scope the terms 'consisting of and 'consisting essentially of features defined in the claimed invention.
  • MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. Rna 2008; 14(11):2348-2360.

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Abstract

La présente invention concerne une méthode de détection du cancer du sein chez un sujet. Plus particulièrement, l'invention concerne une méthode de détection de la présence de microARN exprimés différentiellement dans le sang ou un tissu d'un sujet, lesquels sont caractéristiques de la présence d'un cancer du sein chez le sujet. La présente invention concerne également une méthode de détection si un sujet atteint d'un cancer du sein présente des caractéristiques clinico-pathologiques de positivité ER, positivité Her2 et/ou positivité des ganglions. La présente invention comprend également des trousses destinées à être utilisées dans les méthodes de l'invention.
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WO2016144265A1 (fr) * 2015-03-09 2016-09-15 Agency For Science, Technology And Research Procédé de détermination du risque de développer un cancer du sein par détection des niveaux d'expression de micro-arn (miarn)
WO2016150475A1 (fr) * 2015-03-22 2016-09-29 Universite De Liege Micro-arn circulants pour diagnostiquer le cancer du sein
WO2017031086A1 (fr) * 2015-08-14 2017-02-23 Northwestern University Identification par la plateforme scano-mir d'une signature de microarn circulant distincte pour le diagnostic d'une maladie
WO2019117257A1 (fr) * 2017-12-13 2019-06-20 国立大学法人広島大学 Procédé d'aide à la détection du cancer du sein
WO2023014297A3 (fr) * 2021-08-02 2023-04-20 National University Of Singapore Panel de micro-arn circulants pour la détection précoce du cancer du sein et procédés associés
WO2024127723A1 (fr) * 2022-12-16 2024-06-20 Kabushiki Kaisha Toshiba Procédé d'analyse, kit et dispositif de détection

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016144265A1 (fr) * 2015-03-09 2016-09-15 Agency For Science, Technology And Research Procédé de détermination du risque de développer un cancer du sein par détection des niveaux d'expression de micro-arn (miarn)
WO2016150475A1 (fr) * 2015-03-22 2016-09-29 Universite De Liege Micro-arn circulants pour diagnostiquer le cancer du sein
WO2017031086A1 (fr) * 2015-08-14 2017-02-23 Northwestern University Identification par la plateforme scano-mir d'une signature de microarn circulant distincte pour le diagnostic d'une maladie
WO2019117257A1 (fr) * 2017-12-13 2019-06-20 国立大学法人広島大学 Procédé d'aide à la détection du cancer du sein
JPWO2019117257A1 (ja) * 2017-12-13 2020-12-24 国立大学法人広島大学 乳がんの検出を補助する方法
JP7298913B2 (ja) 2017-12-13 2023-06-27 国立大学法人広島大学 乳がんの検出を補助する方法
WO2023014297A3 (fr) * 2021-08-02 2023-04-20 National University Of Singapore Panel de micro-arn circulants pour la détection précoce du cancer du sein et procédés associés
WO2024127723A1 (fr) * 2022-12-16 2024-06-20 Kabushiki Kaisha Toshiba Procédé d'analyse, kit et dispositif de détection

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