WO2019022605A1

WO2019022605A1 - Micro rna-based methods and compositions for detection of hpv-induced invasive cancers, and their high-grade precursor lesions

Info

Publication number: WO2019022605A1
Application number: PCT/NL2018/050520
Authority: WO
Inventors: Renske Daniëla Maria STEENBERGEN; Christophorus Joannes Lambertus MEIJER
Original assignee: Vrije Universiteit Medisch Centrum VUMC
Current assignee: Vrije Universiteit Medisch Centrum VUMC
Priority date: 2017-07-25
Filing date: 2018-07-25
Publication date: 2019-01-31
Anticipated expiration: 2020-01-25
Also published as: WO2019022605A8

Abstract

The invention relates to the field of cancer prevention and medical diagnostics; and is concerned with a molecular diagnostic assay for cancers, especially human papillomavirus (HPV)-induced invasive cancers and high- grade precursor lesions thereof, such as invasive cervical cancer and premalignant cervical lesions. In particular, the present invention relates to the use of HPV-induced cancer specific miRNA reference genes miR-423, miR-30b, RNU24 and RNU43 to use microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p gene products as markers for hrHPV-induced invasive cancers, and their premalignant lesions with invasive potential.

Description

Title: Micro RNA-based methods and compositions for detection of HPV-induced invasive cancers, and their high-grade precursor lesions

The invention relates to the field of cancer prevention and medical diagnostics; and is concerned with a molecular diagnostic assay for cancers, especially human papillomavirus (HPV)-induced invasive cancers and high- grade precursor lesions thereof, such as invasive cervical cancer and premalignant cervical lesions. In particular, the present invention relates to the use of HPV-induced cancer specific microRNA (miR)-9, -lob, -20a,- 28, - 31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p gene products as markers of which the results are normalised with values from miRNA reference genes miR-423, miR-30b, RNU24 and RNU43 for hrHPV-induced invasive cancers, and their premalignant lesions with invasive potential.

BACKGROUND OF THE INVENTION

Cancer of the uterine cervix is the fourth most common cancer in women world-wide and is responsible for approximately 250.000 cancer deaths a year.

Cervical squamous cell carcinoma development is characterized by a sequence of premalignant lesions, so-called cervical intraepithelial neoplasia (CIN), which are graded 1 to 3, referring to mild dysplasia (CIN 1), moderate dysplasia (CIN 2) and severe dysplasia/carcinoma in situ (CIN 3), respectively. CIN 1 is also referred to as low grade squamous

intraepithelial lesion (LSIL) and CIN 2 and CIN 3 together as high grade squamous intraepithelial lesion (HSIL). For cervical adenocarcinoma, adenocarcinoma in situ (ACIS) is an established precursor lesion. In principle, these premalignant lesions are reversible, although the more severe the lesion, the lower the chance of spontaneous regression. Cervical cancer is considered a preventable disease because the premalignant stages can be detected by exfoliative cytology and treated relatively easily when necessary, with only minor side effects. Cervical screening is aimed to early diagnose the high-grade premalignant (i.e. CIN 2/3 and adenocarcinoma in situ) and treatable cancerous lesions, thereby reducing the mortality of invasive cervical cancer. General medical practice comprises the treatment of all women with morphologically confirmed CIN 2, CIN 3 and

adenocarcinoma in situ, in order to prevent the development of cervical cancer.

Over the past decade it has been well established that cervical carcinogenesis is initiated by an infection with high-risk human

papillomavirus (hrHPV). Expression of the viral oncogenes E6 and E7, which disturb the p53 and Rb tumor suppressor pathways, respectively, has been shown to be essential for both the onset of oncogenesis and the maintenance of a malignant phenotype. Therefore, testing for hrHPV appeared an attractive, primary screening tool. However, consistent with a multistep process of carcinogenesis, additional alterations in the host cell genome are required for progression of an hrHPV infected cell to invasive cancer cell. Only a small proportion of women infected with high-risk HPV will develop high-grade premalignant cervical lesions (CIN 2/3) and, if left untreated, cervical cancer. In most women with premalignant cervical lesions the lesions regress spontaneously. Of the women who participate in population based screening, about 5-6% have a positive hrHPV test.

However, only at maximum 20% of them (1% of the participating women) have >CIN 2/3. Therefore, primary screening by hrHPV testing will be accompanied with a substantial number of redundant follow-up procedures and unnecessary anxiety amongst women, unless markers can be applied to the cervical smears that allow stratification of hrHPV positive women for risk of >CIN 2/3 and >adenocarcinoma in situ.

A major challenge is to reduce the percentage of HPV positive women to those that have clinically meaningful lesions. One mode is to use cytology as a secondary (so-called triage) test for hrHPV positive women. Still, this leaves a substantial number of hrHPV positive women with normal cytology (3.5% of the women in the screening population), of which still 10% have or acquire >CIN 3. Moreover, cytology is not an option for self-sampled cervico-vaginal specimens that can be taken at home, since these are not representative for the cytologicai status of the cervix. Another mode is to use HPV16/18 genotyping. This however leaves women with non- HPV16/18 types who are, although to a lesser extent, also at risk of >CIN 2/3 and ^adenocarcinoma in situ. Therefore, there is a need for

supplementary or alternative triage tools to stratify hrHPV positive women into those with and without >CIN 2/3 and ^adenocarcinoma in situ.

Primary screening for cervical cancer using disease markers based on host cell changes in cancer genes provides a promising alternative provided that specificity and sensitivity is sufficiently high. This option is of particular interest for low and middle income countries, where quality - controlled cytology is absent and implementation of follow-up algorithms for HPV-positive women is complicated. In these countries self- sampling has shown to facilitate access to cervical screening (Laczano-Ponce et al., Lancet. 2011; 378: 1868-1873). In this sense it is extremely useful to have markers that also prevail in self-samples. It appears that there is a huge difference in the 'behaviour' of markers on vaginal smears that have been obtained by medical skilled personnel like doctors or nurses and on vaginal swabs that have been collected by the woman herself. It has appeared that many markers that would be suitable for doctor-provided samples are not useful in self-samples. The necessity to work with self-samples instead of doctor samples is high in low and middle income countries since in those countries there is less medical personnel per capita and often there is a cultural problem by letting other persons taking a vaginal sample. Further, even in highly developed countries self- sampling is an ideal way to reduce the costs of obtaining vaginal samples for large scale cervical cancer screening. An even higher acceptance rate can be expected when urine samples can be used for early cancer detection.

MicroRNAs are a class of small, non-coding RNAs that control gene expression by hybridizing to and triggering either translational repression or, less frequently, degradation of a messenger RNA (mRNA) target. The discovery and study of miRNAs has revealed miRNA-mediated gene regulatory mechanisms that play important roles in organismal

development and various cellular processes, such as cell differentiation, cell growth and cell death (Cheng et al, Nucleic Acids Res. 33:1290-1297 (2005)). Recent studies suggest that aberrant expression of particular miRNAs may be involved in human diseases, such as cancer (Li and Kowdley, Genomics Proteomics Bioinformatics 10: 246-253 (2012); Lee, J.-W. et al., Clin. Cancer Res. 2008, 14(9):2535-2542). Since then, a number of studies has been published that show that aberrant expression of miRNAs also occurs with HPV-induced lesions and carcinomas (He, Y. et al., Int. J. Cancer, 2016, 15;138(6):1312-27; Wang, X. et al., Proc. Natl. Acad. Sci. USA 2014, lll(ll):4262-4267; Wilting, S.M. et al, Oncogene 2013, 32(1):106-116;

Satapathy, S. et al., Expert Rev. Mol. Diagn. 2017, 17(7):711-722).

However, for reliable and reproducible miRNA expression analysis adequate data normalization is essential to remove non-biological, technical variations introduced during the experimental procedure. We have found that relevant and adequate reference genes differ between different types of specimens, even when originating from the same anatomical source.

The present invention provides novel methods and compositions for the diagnosis and treatments of hrHPV-induced invasive cancers and their premalignant lesions with invasive potential using cervical exfoliated cells, cervico-vaginal self-collected specimens and urine. Additionally, the present invention provides relevant reference genes for microRNA quantitative PCR analysis and its application to cervical specimens. SUMMARY OF THE INVENTION

The inventors now have found relevant and adequate RNA reference gene products for normalising microRNA gene product levels in order to remove non-biological, technical variations when analysing miRNA expression in cervical specimens. In a preferred embodiment of the invention the level of gene products of reference gene miR-423 in

combination with RNU24 or RNU43 are relevant for normalisation of miRNA gene product levels obtained from cervical scraping or smears.

In another preferred embodiment of the invention the level of gene products of reference gene miR-423 in combination with miR30b are relevant for normalisation of miRNA gene product levels obtained from self- collected cervico-vaginal specimens.

Next to that the inventors have found a method for detecting HPV-induced invasive cancers, and their high-grade precancerous lesions wherein said method comprises the detection of at least one microRNA gene product selected from the group of (miR)-9, -15b, -20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p normalized to at least one

(sampling type specific) RNA reference gene product selected from the group of miR-423, miR-30b, RNU24 and RNU43 in a cervical specimen whereby the presence of such said gene product normalized for said (sampling type specific) reference gene product indicates the presence of HPV-induced precursor lesions with invasive potential and/or HPV-induced invasive cancers.

Preferably, in such a method said HPV-induced high-grade precancerous lesion or HPV-induced invasive carcinoma is a high-grade premalignant cervical lesion or invasive cervical cancer, more preferably a high-risk HPV-induced invasive cancer.

In a preferred embodiment of the invention the level of at least one of microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, - 222, -375 and let-7b-5p gene product for cervical scrapings normalized for at least a combination of the miR-423 in combination with RNU24 or RNU 43 gene product in smears, or in self-collected cervico-vaginal specimens normalized for at least a combination of the miR-423 with miR-30b gene product, is detected.

Next to that the inventors have found a method for detecting HPV-induced invasive cancers, and their high-grade precancerous lesions wherein said method comprises the detection of at least one microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let- 7b-5p gene product in urine and/or in small vesicles isolated from urine whereby such said gene product indicates the presence of HPV-induced precursor lesions with invasive potential and/or, HPV-induced invasive carcinoma . Preferably, in such a method said HPV-induced high-grade precancerous lesion or HPV-induced invasive carcinoma is a high-grade premalignant cervical lesion or invasive cervical cancer, more preferably a high-risk HPV-induced invasive cancer.

The invention also relates to a method as defined above wherein altered expression is detected of microRNA (miR)-9, -15b, -20a,- 28, -31, -93, - 100,- 125b, -149, -203, -222, -375 and/or let-7b-5p normalized to miRNA reference genes miR-423, miR-30b, RNU24 and RNU43 in the test cell as compared to the comparable normal cell.

Also part of the invention is the use of microRNA (miR)-9, -15b,- 20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let- 7b- 5p gene product preferably normalized to miRNA reference genes miR-423, miR-30b, RNU24 and/or RNU43 as a molecular diagnostic marker for the detection of HPV-induced invasive carcinoma, and their high-grade precancerous lesion. Preferably, in such a use the gene product of said markers is predictive for the occurrence of said lesion or carcinoma. The invention also comprises a kit of parts for use in a method of detecting HPV-induced invasive carcinoma, and their high-grade

precancerous lesion said kit comprising

-means for the detection of miRNA reference genes miR-423, miR- 30b, RNU24 and/or RNU43 and microRNA (miR)-9, -15b,-20a,- 28, -31, -93, - 100,- 125b, -149, -203, -222, -375 and let-7b-5p gene products

-means for the detection of HPV infection, wherein said means comprise probes and primers specific for HPV.

The invention further relates to a method for detecting HPV- induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- measuring in a cell derived from a sample from a subject suspected having, or running a risk in developing said lesions or carcinomas, the expression of the product of any classifier comprised of two or more of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let- 7b-5p genes, preferably wherein said classifier is composed of the two miRNAs miR-15b and miR-375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let-7b, miR-15b, miR-20a, miR-93, and miR-222;

- normalising said expression levels on basis of the expression of the reference genes miR-423 in combination with miR30b, RNU43 or RNU24 and;

- comparing said levels with the levels of healthy controls,

wherein said sample is determined as positive if the normalised expression levels of the classifier deviate from the control normalised expression levels.

Also further comprised in the invention is a kit of parts for use in a method of detecting HPV-induced high- grade precancerous lesion or HPV- induced invasive carcinoma or nonHPV-induced gynaecological or

anogenital cancer, said kit comprising means for the detection of HPV infection, wherein said means comprise probes and primers specific for hrHPV virus and wherein said kit further comprises means for the detection of expression of any classifier comprised of two or more of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let- 7b-5p genes, preferably wherein said classifier is composed of the two miRNAs miR-15b and miR-375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let-7b, miR-15b, miR-20a, miR-93, and miR-222and optionally means for the detection of expression of the product of the reference gene miR-423 and one or more of miR30b, RNU43 and RNU24.

Also part of the invention is a method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- measuring in a cell derived from a sample from a subject suspected having, or running a risk in developing said lesions or carcinomas, the expression one or more of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, - 149, -203, -222, -375 and let-7b-5p or the expression of the product of any classifier comprised of two or more of the microRNA (miR)-9, -15b, -20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes preferably wherein said classifier is composed of the two miRNAs miR-15b and miR- 375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let- 7b, miR-15b, miR-20a, miR-93, and miR-222;

- measuring in said cell the degree of methylation of methylation marker FAM19A4,

- comparing said levels with the levels of healthy controls,

wherein said sample is determined as positive if the normalised expression levels of the classifier and/or of the methylation marker deviate from the control normalised expression levels The level of the (at least one) miRNA reference gene product and the (at least one) miRNA gene product can be measured using a variety of techniques that are well known to those of skill in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection, nanotechnology (e.g. lab on a chip), etc. An alteration in the signal of at least one normalized miRNA gene product in the test sample relative to control samples is indicative of the subject either having, or being at risk for developing HPV-induced invasive carcinoma, nonHPV-induced

gynaecological or anogenital carcinoma and their high- grade precancerous lesion.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Flow scheme of strategy used for the selection of reference genes for miRNA qRT-PCR normalization.

Figure 2. Analysis of p-values between normal, CIN2/3 and SCC cervical specimens. No normalization, normalization using 2 commonly used reference genes (U6+RNU6B) and the 2 biologically most stable reference genes (tissues: RNU24+miR-423, scrapes: RNU43+miR-423, self-samples: miR-423+miR-30b) was evaluated. P-values were computed by a two-sample t-statistic on log2 transformed expression data for (A) miR-15b and (B) miR-100. The black line indicates significance level 0.05. Positive signal-to- noise ratios indicate upregulation , negative ratios correspond to

do wnre gulation .

Figure 3 shows the boxplots of the miRNA expression levels as measured by RT qPCR in HPV-positive cervical scrapes of women with cervical cancer (SCC and AC), CIN3 and controls. On the y-axes levels of miRNA expression are presented; on the x-axes groups represent the patient cohorts tested. Boxplots show medians with upper and lower quartile, and range whiskers. * P < 0.05, ** P < 0.01, *** P < 0.001. The levels of miRNA- 9, -15b, 125b, -149, and -375 expression when normalized to miR-423 and RNU24 expression are significantly altered in women with SCC, women with AC and women with CIN3 compared to controls.

Figure 4 shows the boxplots of the miRNA expression levels as measured by RT qPCR in HPV-positive self-collected cervicovaginal specimens of women with squamous cervical cancer, CIN3 and controls. On the y-axes levels of miRNA expression are presented; on the x-axes groups represent the patient cohorts tested. Boxplots show medians with upper and lower quartile, and range whiskers. * P < 0.05, ** P < 0.01, *** P < 0.001. The levels of let-7b-5p, miRNA-15b, -20a, -31, -93 and -222 are significantly altered in women with cervical cancer and CIN3 compared to controls.

Figure 5 shows the boxplots of the miRNA expression levels as measured by RT qPCR in extracellular vehicles (exosomes) isolated from urine of women with squamous cervical cancer (n=ll) and healthy controls (n=ll). On the y-axes levels of miRNA expression are presented; on the x- axes groups represent control women and women with cervical cancer. Boxplots show medians with upper and lower quartile, and range whiskers. The levels of miRNA-9, -15b, -100, -125b, -203a, -375 and -21 are

significantly altered in women with cervical cancer compared to controls. The ratio of miR-21 over miR-9 shows highest discriminatory power (bottom right).

Fig. 6 ROC curve analysis of HPV16/18 genotyping and the 2- miRNA classifier for the detection of CIN3. Results obtained from scrapes with known HPV16/18 genotyping results (65 normal, 108 CIN3) were used to build classifiers for HPV16/18 genotyping (HPV), a new 2-miRNA classifier (miR-15b/375) and the 2-miRNA classifier combined with

HPV16/18 genotyping (miR-15b/375/HPV). Classifiers were validated by leave-one-out cross-validation. The model miR-15b/375/HPV is significantly better than the 2-miRNA classifier (p = 0.011) Figure 7: Performance of the 3-miRNA classifier for the detection of cervical disease (CIN, cervical intraepithelial neoplasia; SCC, squamous cell carcinoma; AC, adenocarcinoma).

A) Results obtained from 77 hrHPV-positive scrapes from women without underlying disease (CINO/1) and 55 scrapes from women with

CIN3/AIS were used to build a 3-miRNA classifier for the detection of CIN3. The classifier was validated by leave-one-out cross-validation. The diagonal line indicates an AUC of 0.5.

B) Predicted probabilities obtained for all samples using the 3- miRNA classifier (77 CINO/1, 48 CIN2, 55 CIN3, 25 SCC, 5 AQ.AUC, area under the curve.

Figure 8. miRNAs are complementary to methylation marker FAM19A4. Overview of samples positive for 3-miRNA classifier, FAM19A4 methylation, the 3-miRNA classifier AND/OR FAM19A4 and the 3-miRNA classifier AND FAM19A4 methylation. Samples are sorted based on their 3- miRNA classifier predicted probability and histology (CIN, cervical intraepithelial neoplasia; AIS, adenocarcinoma in-situ; SCC, squamous cell carcinoma; AC, adenocarcinoma)..

Figure 9: Clinical performance and expression levels of the miRNA markers in HPV-positive self-samples. (A) ROC curve and AUC of a 5-miRNA marker panel (let- 7b, miR-15b, miR-20a, miR-93, and miR-222 in HPV-positive self-samples for CIN3+ detection (101 controls, 48 CIN3, 41 SCC). (B) ROC curve and AUC of the 6 miRNAs in HPV-positive self-samples (01 controls, 48 CIN3, 41 SCC). For comparison, the ROC curve and AUC of the 5- miRNA marker panel is included. DETAILED DESCRIPTION OF THE INVENTION

The term "HPV-induced invasive cancer" refers to a carcinoma induced by high-risk HPV, which invades surrounding tissue. This includes all HPV-induced carcinoma histotypes, i.e., squamous cell carcinomas (SCC), adenocarcinomas, adenosquamous carcinomas (AC) and

neuroendocrine carcinomas in relevant organs such as cervix, oral cavity, oropharynx, anus, rectum, penis, vulva, vagina, etc. It especially includes head and neck squamous cell carcinomas (HNSCC), cervical squamous cell carcinomas and cervical adenocarcinomas.

The term "invasive cervical cancer" refers to a cervical carcinoma invading surrounding tissue. This includes all carcinoma histotypes, i.e., squamous cell carcinomas, adenocarcinomas, adenosquamous cell carcinomas and neuroendocrine carcinomas.

The terms "premalignant lesion" and "precursor lesion" refer to a stage in the multistep cellular evolution to cancer with a strongly increased chance to progress to a carcinoma. With classical morphology the

pathologist is unable to predict in the individual patient which of these lesions will progress or regress. The current patent refers to a method, which can predict invasive cancer or a high-grade precursor lesion thereof.

The term "high-grade premalignant cervical lesion" refers to a stage in the multistep cellular evolution to cervical cancer with a strongly increased chance to progress to a cervical carcinoma. The term "capable of specifically hybridizing to" refers to a nucleic acid sequence capable of specific base-pairing with a complementary nucleic acid sequence and binding thereto to form a nucleic acid duplex.

A "complement" or "complementary sequence" is a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-paring rules. For example, the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'. The term "stringent hybridization conditions" refers to

hybridization conditions that affect the stability of hybrids, e.g.,

temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of the primer or the probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O.lx SSC at 60°C. Hybridization procedures are well known in the art and are described in e.g. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994.

The term "oligonucleotide" refers to a short sequence of nucleotide monomers (usually 6 to 100 nucleotides) joined by phosphorous linkages (e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), or non- phosphorous linkages (e.g., peptide, sulfamate and others). An

oligonucleotide may contain modified nucleotides having modified bases (e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2'-O-methyl ribosyl, 2'-0-methoxyethyl ribosyl, 2'-fluoro ribosyl, 2'-amino ribosyl, and the like). Oligonucleotides may be naturally-occurring or synthetic molecules of double- and single-stranded DNA and double- and single- stranded RNA with circular, branched or linear shapes and optionally including domains capable of forming stable secondary structures (e.g., stem-and-loop and loop- stem-loop structures).

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA

polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The

(amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxy ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. A "pair of bi-directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in polymerase chain reaction (PCR) amplification.

The term "probe" refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that control gene expression by hybridizing to and triggering either translational repression or, less frequently, degradation of a messenger RNA (mRNA) target. miRNA-mediated gene regulatory mechanisms play important roles in organismal development and various cellular processes, such as cell differentiation, cell growth and cell death (Cheng et al, Nucleic Acids Res. 33:1290-1297 (2005)). Recent studies suggest that aberrant expression of particular miRNAs may be involved in human diseases, such as cancer (Li and Kowdley, Genomics Proteomics Bioinformatics 10: 246—253 (2012)).

In order to perform reliable miRNA expression analysis using qRT- PCR, suitable and robust data normalization is recommended to remove or minimize non-biological sample-to-sample variations introduced during the experimental procedure. We successfully identified miR-423 as a suitable reference gene for cervical scrapes, and self-collected sample types, to be used in combination with RNU43 or RNU24 in cervical scrapes and with miR-30b in self-samples. Our strategy used for the identification of relevant and adequate reference genes is shown in Figure 1, and the use of these normalizers increases the signal-to-noise ratio which is not observed when normalizing to commonly employed reference genes (e.g. U6 and RNU6B). MicroRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p targets are important determinants of hr-HPV induced carcinogenesis, as has been described in the literature cited in the

background part of the present application. MicroRNA (miR)-9, -15b, -20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene products normalized to miR-423, mlR-30b, RNU24 and/or RNU43 thus provide valuable markers to diagnose invasive cervical cancer and the high-grade precursor lesions thereof in different sampling types, i.e. cervical tissues, cervical scrapes, self-collected cervico-vaginal specimens, as well as urine specimens en exosomes isolated thereof as is shown in the present application.

For normalization on basis of two or more reference genes the geometric mean of the expression level of said two or more reference genes is calculated. Relative miRNA expression is then determined using the

method (Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402-408). It appears that with the use of the miR-423, miR-30b, RNU24 and/or RNU43 gene products an adequate normalization can be achieved and moreover, that this normalization, i.e. the combination of miRNA gene products that optimally need to be used for normalization, is sample specific. As is shown in the experimental part, normalization with the combination of miR-423 and miR-30b is ideally used for cervical self- samples, while normalization with the combination of miR-423 with either or both of RNU24 and RNU43 is ideally used for cervical scrapes.

Cervical cancer is almost exclusively associated with human papillomavirus (HPV) infection. Human papillomaviruses, constitute a group of more than 150 types of viruses, as identified by variations in DNA sequence. The various HPVs cause a variety of cutaneous and mucosal diseases. HPVs are broadly classified into low-risk and high-risk types, based on their ability to induce malignant changes in infected cells. Low risk HPV types such as 1, 2, 4, 6, 11, 13 and 32 are primarily associated with benign lesions or common warts, while the high risk types, such as 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 are primarily associated with premalignant and malignant epithelial lesions. The high-risk HPV types have been found to cause invasive carcinoma of the uterine cervix, as well as invasive carcinoma elsewhere in the anogenital tract and/or head- neck region. Therefore, the present invention is not only suited to detect invasive cervical cancer and precursor stages thereof , but also other invasive cancers and corresponding precursor stages that are induced by HPV, particularly of the high-risk type. Thus, the present invention provides a method for the risk assessment of any HPV-induced high-grade premalignant lesion or invasive cancer.

Very suitable HPV-induced precursor lesions and invasive cancers in the context of the present invention are cervical precancerous lesions and invasive cervical cancers, but also precursor lesions and invasive cancers induced by high-risk HPV in other tissues such as oral cavity, oropharynx, anus, rectum, penis, vulva, vagina, etc.

A test cell may be a (pre)neoplastic cell, a proliferating cervical cell, or any other cell wherein the presence of an HPV-induced precursor lesion with invasive potential, HPV-induced invasive cancer, is to be detected. Said cell is preferably obtained from a subject in which HPV infection with one of the hrHPV types as provided above, has already been established

Although correlations between microRNA (miR)-9, -15b, -20a,- 28, - 31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p gene product levels and cervical cancer have been described, their diagnostic value after normalisation for sampling type-specific reference genes to detect cervical cancer and CIN3 upon testing cervical material is novel. Importantly, virtually all women with cervical cancer showed altered normalized miR gene product levels independent of the sample type tested.

More importantly, the detection of altered miRNA expression for the diagnosis of cervical cancer in urine and exosomes isolated thereof has not been described before. It is surprising that the detection of these miRNAs in urine is possible and still provides for an adequate detection of pre-cancerous lesions and/or cervical cancer. This finding, of course, offers a major advantage in the diagnostic use of HPV related urogenital cancers, since it is easier and less invasive to obtain a urine sample and providing a urine sample for diagnostic purposes is well accepted and routinely performed, both in hospitalized patients, but also in ambulant, non- hospitalized women.

The present inventors have now established that detection of miR- 423, miR-30b, RNU24 and/or RNU43 reference genes and microRNA (miR)- 9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene product levels in physician-collected cervical scrapes, self-collected cervico-vaginal specimens and urine and extracellular vesicle derived thereof is able to predict the presence of a high-grade CIN lesion or invasive carcinoma.

Accordingly, the present invention provides a method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive cancers, said method comprising detection of miR-423, miR-30b, RNU24 and/or RNU43 reference genes and microRNA (miR)-9, -15b, -20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene product levels in a cell or cell product whereby such gene product levels indicates the presence of HPV-induced precursor lesions with invasive potential and HPV-induced invasive cancers The test cell of the subject may comprise a cell or cell product from a sample of mucosal cells, such as cervical cells, and other tissues wherein a precursor lesion or cancer associated with HPV is to be detected. All such samples may be used as a sample in a method of the present invention. Preferably, a sample of a patient's cells comprise cervical cells or other epithelial cells of the anogenital or oropharyngeal tract as test cells. The cervical cells may e.g. be presented as cytological specimen.

Cytological specimens comprise conventional cervical smears as well as thin layer preparations of cervical specimens and cervico-vaginal or vaginal specimens collected by self- sampling. Alternatively, cells or cell products, such as small vesicles, may be presented in urine samples. A test cell wherein the present invention is especially advantageous over other known methods of detecting cancers in the cervix and adjacent tissues is a test cell obtained from a self-sample or a urine sample.

A method of the present invention is particularly suited for the detection of high-grade precancerous lesions and invasive cancers associated with microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, - 222, -375 and/or let-7b-5p gene levels normalized to miR-423, miR-30b, RNU24 and/or RNU43 reference genes that are induced by high-risk HPVs or derived from the (female) anogenital tract. A method of detecting HPV- induced high-grade precancerous lesions with invasive potential, HPV- induced invasive cancers may comprise measuring microRNA (miR)-9, -15b,- 20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let- 7b- 5p gene levels normalized to miR-423, miR-30b, RNU24 and RNU43 reference genes

The test cell component may be detected directly in situ or it may be isolated from other cell components by common methods known to those of skill in the art before contacting with the reagent (see for example,

"Current Protocols in Molecular Biology", Ausubel et al. 1995. 4th edition, John Wiley and Sons; "A Laboratory Guide to RNA: Isolation, analysis, and synthesis", Krieg (ed.), 1996, Wiley-Liss; "Molecular Cloning: A laboratory manual", J. Sambrook, E.F. Fritsch. 1989. 3 Vols, 2nd edition, Cold Spring Harbor Laboratory Press)

Any specimen containing a detectable amount of miR-423, miR- 30b, RNU24 and/or RNU43 and microRNA (miR)-9, -15b,-20a,- 28, -31, -93, - 100,- 125b, -149, -203, -222, -375 and/or let-7b-5p polynucleotide can be used. Preferred samples for testing according to methods of the invention include such specimens as (cervical or vaginal) scrapes, cervico-vaginal lavages or swabs, urine, blood and/or (cervical) biopsies and the like.

Although the subject can be any mammal, preferably the subject is human.

Specifically for detection of said lesions or carcinomas in urine the detection of the expression of miR-9 when normalized to miR-423 shows excellent results. Alternatively, another very well performing parameter for the detection of said lesions or carcinomas is the parameter that constitutes the ratio of expression of miR-21 over expression of miR-9. In the latter case no normalisation is needed, because the influence of the measurement errors is neutralised by taking the ratio between the two miRNAs.

Diagnostic methods for the detection of disorders_T include methods wherein a sample for testing is provided, which sample comprises a cell preparation from cervical or other tissue. Preferably such samples are provided as cytological samples or urine. Additional suitable samples include blood and tissue biopsies.

A cell or tissue sample obtained from a mammal, preferably a human, is suitably pre-treated to allow contact between the cellular RNA of a test cell comprised in said sample with a reagent that detects an

alteration in microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, - 203, -222, -375 and/or let-7b-5p gene product levels normalized to miR-423, miR-30b, RNU24 and/or RNU43 as compared to that of a comparable normal cell. Samples may be mounted on a suitable support to allow observation of individual cells. Examples of well-known support materials include glass, polystyrene, polypropylene, polyethylene, polycarbonate, polyurethane, optionally provided with layers to improve cell adhesion and immobilization of the sample, such as layers of poly-L-lysine or silane.

Cervical smears or biopsies may for instance be prepared as for the

Papanicolaou (Pap) test or any suitable modification thereof as known by the skilled person, and may be fixed by procedures that allow proper access of the reagent to the target component. In certain embodiments of the invention the cytological specimens are provided as conventional smear samples or thin layer preparations of cervical cells or liquid based cytology samples or any other kind of preparation known to those of skill in the art. If storage is required, routine procedures use buffered formalin for fixation followed by paraffin embedding, which provides for a well-preserved tissue infrastructure. In one embodiment of a method of the invention miR-423, miR- 30b, RNU24 and/or RNU43 reference genes and microRNA (miR)-9, -15b,- 20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene product levels in the test cell or cell product are detected as compared to the comparable normal cell or cell product.

It may also be that detection of one or more of these miRNAs is performed in connection with the detection of a methylation marker, such as FAM19A4. Methods for performing assays with methylation marker

FAM19A4 have been described previously (see e.g. WO 2014/058321). It is believed that in many cases the detection of one or more miRNAs and the detection of FAM19A4 are complimentary thereby increasing the sensitivity of such an assay.

Further, it appeared that a combination of two or more of the miRNA expression markers would lead to an improved detection of HPV- induced high-grade precancerous lesions and HPV-induced invasive cancers. These combinations are generally indicated as a 'classifier'. For cervical scrapes it appeared that a 2-miRNA combining miR-15b and miR-375 (normalized by miR-423 and RNU24) classifier outperformed detection by individual miRNA expression. In this case, it was found that expression of miR-9b was upregulated in scrapes of patients, while expression of miR-375 was downregulated.

In a further study (see Example 6) it was found that a 3-miRNA classifier performed optimal and even better than the above-mentioned 2- miRNA classifier. This optimal miRNA classifier consisted of miR-149, miR- 20a and miR-93 (again normalised by miR-423 and RNU24). At 70% specificity, this 3-miRNA classifier detected 76% of CIN3. Moreover, the same classifier applied in the remaining samples detected 60% of CIN2 lesions, all SCC and all AC, and therefore showed a clear improvement in performance compared to the individual miRNAs. In total, a CIN3+ detection rate of 85% was achieved. It further appeared that addition of more miRNAs to the classifier did not improve the performance further.

It further appeared in this study (see Example 7) that such a classifier can be ideally combined with detection of HPV- induced

precancerous lesions and invasive cancers with methylation analysis of a further marker, FAM19A4. A combined model consisting of miR-149, let-7b, miR-20a, miR-93 and FAM19A4 achieved a CIN3 detection rate of 93% at 70% specificity. This combined miRNA/FAM 19A4 classifier (4 miRNAs + FAM19A4) was significantly better than the 3-miRNA classifier and

FAM19A4 alone.

Also for self-samples a classifier may be used. In the experiment reported in Example 8 it appeared that a 5-miRNA signature consisting of let-7b, miR-15b, miR-20a, miR-93, and miR-222 (AUC=0.78) resulted in detection with a sensitivity of 67% and a specificity of 65% for CIN3 detection, which was higher tha of the individual miRNAs or any other combination. Importantly, 93% (38 out of 41) of the cervical cancers were detected.

For comparison of the classifier with normal controls the sum of the differences of each of the expression levels of the miRNAs that form the classifier with the corresponding control expression level is taken and the sum of these accumulated differences forms the decisive value for the detection.

Accordingly, the present invention also provides for a method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- comparing said levels with the levels of healthy controls,

The present invention also provides a kit of parts as defined in the claims, for use in a method of detecting HPV-induced precursor lesions with invasive potential, HPV-induced invasive cancers . Such a kit may suitably comprise a brush or spatula to take a (cervical) scrape either or not together with a container filled with collection medium to collect test cells.

Alternatively, a sampling device consisting of an irrigation syringe, a disposable female urine catheter and a container with irrigation fluid will be included to collect cervical cells by cervico-vaginal lavage. Additionally, a container to collect urine is suitable, preferably to be used to collect first- void urine. A kit according to the present invention further comprises primers and probes for the detection of miR-423, miR-30b, RNU24 and/or RNU43 reference genes and microRNA (miR)-9, -15b,-20a,- 28, -31, -93, - 100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene product level.

In yet another alternative embodiment of a kit of the invention the means for the detection of miR-423, miR-30b, RNU24 and/or RNU43 reference genes and microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and/or let-7b-5p gene product levels may be combined with means for the detection of HPV infection, preferably for the detection of HPV infection of the high-risk type. Such means may comprise HPV-specific primers or probes, protein markers for HPV infection or even surrogate markers for HPV infection as are known in the art. Preferably, the means for detection of HPV infection are means for the detection of HPV16/18 genotyping, where this MHPV16/18 genotyping and miRNA expression analysis can be performed on the same cervical scrape.

Combination of the method of the present invention with HPV16/18 can further improve the detection of cervical precancerous lesions as shown in the Examples below.

The present invention will now be illustrated by way of the following, non limiting examples. EXAMPLES

Example 1. Strategy used to find relevant reference genes for microRNA qPCR analysis in cervical specimens.

In a comprehensive analysis the performance of 10 candidate reference genes in three different types of cervical specimens, i.e. tissues , scrapes and self-collected cervicovaginal specimens (self- samples) obtained from women with varying degrees of cervical disease, has been conducted according to the our developed multi-step selection strategy shown in Figure 1.

The 10 reference genes included RNU24, RNU43, RNU6B, U75, miR-30b, miR-378i, miR-423, miR-425, miR-605 and miR-631, and were selected as candidate reference genes based on literature (Vandecompele J., et al., Genome Biol. 2002, 3:research0034.1-research0034.11; Bengtsson, M. et al., BMC Mol. Biol. 2008, 9:263; Korenkova, V. et al, BMC Mol. Biol. 2015, 16:5) and in-house available genome-wide miRNA profiles of women with and without (pre)cancerous disease (see Example 3). Expression levels of the 10 candidate reference genes were determined using miRNA- specific TaqMan assays. Three commonly employed algorithms GeNorm, NormFinder and BestKeeper were combined to identify the biologically most stable reference genes for normalization of miRNA qRT-PCR data in each specific sample type. We identified miR-423 as a suitable reference gene for all sample types, to be used in combination with RNU24 or RNU43 in cervical scrapes and miR- 30b in self- samples. The findings demonstrate that the choice of reference genes differs between different types of specimens, even when originating from the same anatomical source.

As demonstrated for two miRNAs associated with cervical cancer, miRl5b and miR-100, normalization with these suitable reference genes for the specific sample types increased the signal-to-noise ratio and reduced the p- value, which was not observed when normalizing to commonly employed reference genes U6 and RNU6B (Figure 2; p-values). A larger signal-to-noise ratio and a smaller p-value indicates that the technical noise is compressed whereas the biological differences are not (or hardly) affected by

normalization. Example 2.

A comprehensive analysis of normalized miR gene product levels changes detectable in cervical scrapes a has been conducted by means of quantitative reverse transcriptase PCR on hrHPV-positive cervical samples. The cervical scrapes included 66 hrHPV-positive samples of women without cervical disease, 121 of women with CIN3, 29 of women with SCC and 9 of women with AC. As shown in Figure 3 miRNA-9, -15b, 125b, -149, and -375 expression normalized to miR-423 and RNU24 expression was significantly different between HPV+ control women and women with CIN3, SCC and AC. Importantly the combination of miR-15b and miR-375 normalized to the geometric mean of miR-423 and RNU24 detected all cancers.

Example 3

To identify miRNAs detectable in self- samples that can predict the presence of CIN3 and cervical cancer we set out to determine, for the first time, genome-wide miRNA profiles in hrHPV-positive self-collected cervico- vaginal specimens. Small RNA sequencing (RNA-Seq) was conducted to determine genome-wide miRNA expression profiles in hrHPV-positive self- collected cervico-vaginal specimens (36 of women without cervical disease during follow-up (<CIN1), and 37 of women with CIN3 lesions). Candidate miRNA markers, i.e. let-7b-5p, miRNA-15b, -20a, -31, -93 and -222, were validated by means of quantitative reverse transcriptase PCR on hrHPV- positive self-collected cervico-vaginal specimens. The self-collected cervico- vaginal specimens included 101 hrHPV-positive samples of women without cervical disease, 48 of women with CIN3 and 15 of women with SCC. As shown in Figure 4 let-7b-5p, miRNA-15b, -20a, -31, -93 and -222 expression normalized to miR-423 and miR-30b expression was significantly different between HPV+ control women and women with CIN3, and SCC.

These data show that CIN3 and cervical cancer associated miRNAs can be detected in hrHPV-positive self-samples.

Example 4

To assess whether differential miRNA expression analysis in urine, in particular in extracellular vesicles isolated from urine, can predict the presence of cervical cancer, we isolated extracellular vesicles (exosomes) from urine of 11 control women and 11 women with SCC. As shown in

Figure 5 the miRNA expression levels of miRNA-9, -15b, -100, -125b, -203a and -375 are lower in women with SCC compared to controls. Even in this relatively small sample group, significant differences (P<0.05) were shown for miR-9, miR-125b, miR203a. Analysis of the ratio of miR-21 over miR-9 and miR-9 over miR-423 revealed an even higher discriminatory power (P^O.007 and P0.004 respectively). These data show for the first time that measuring of expression of microRNAs in urine can be used to detect cervical cancer. Example 5

In the study described in Example 2 it was further assessed whether HPV16/18 genotyping would improve the detection of CIN3 and cancer. For this, samples were either classified as HPV16/18 positive (19 normal, 71 CIN3) or other hrHPV type positive (46 normal, 37 CIN3), for those samples for which the genotype was known (n = 173 out of 187). For this smaller sample set, we built (i) a new 2-miRNA classifier consisting of miR-15b and miR-375 and (ii) a logistic regression model combining the 2-miRNA classifier with HPV16/18 genotyping. Consistent with previous reports, HPV16/18 genotyping achieved 66% sensitivity and 68% specificity for CIN3 detection. The 2-miRNA classifier obtained in the smaller sample set had a comparable performance to the one obtained from the entire set of samples. While HPV16/18 genotyping alone was inferior to the 2-miRNA classifier (p = 5.2e-16, Fig. 6), a classifier combining our two selected miRNAs with HPV16/18 genotyping had an improved performance and achieved an AUC of 0.712 (Table 1, Fig. 6). The 2-miRNA classifier adjusted by HPV16/18 type had a significantly better performance than the 2-miRNA classifier (p = 0.011). Including HPV16/18 genotyping in the classifier increased both sensitivity and specificity to 63 and 77%, respectively, and all SCC and AC were detected.

Table 1 Optimal sensitivity and specificity for the detection of CIN3 for hrHPV type (HPV16/18, others) and the miRNA classifier in conjunction with hrHPV type based on a smaller sample set with known hrHPV type infection and leave-one-out cross-validation

Example 6 Expression of let-7b, iR-15b-, miR-125b-, miR-149-, miR-20a, miR

203a-, miR-222, miR-31, miR 375 and miR-93 was measured using TaqMan microRNA assays in a sample set of 210 hrHPV-positive cervical scrapes. RNU24 and miR-423 were included as reference genes. Multi-miRNA classifiers for the detection of CIN3 were built performing multivariable logistic regression followed by backward elimination on square root transformed delta Ct ratios and evaluated using leave-one-out cross- validation. As a result, predicted probabilities, i.e. values between 0 and 1 representing the risk of an underlying CIN3, were calculated for each sample. Receiver-operated characteristic (ROC) curve analysis was carried out to evaluate the performance of the miRNA classifiers in detecting CIN3.

At 70% specificity, individual miRNAs detected between 33% (miR- 15b) and 60% (miR-149) of CIN3. Unfortunately, none of the tested miRNAs achieved a SCC detection rate of 100%. miR-375 alone, however, detected 92% of SCC, while miR-149 and miR-203a detected all AC. The highest CIN3+ detection rate was obtained by miR-149 (68%). See Table 2. There was no loss in model performance when miRNA markers were excluded using backward elimination. The final optimal miRNA classifier consisted of miR-149, miR-20a and miR-93 and achieved an AUC of 0.808 (Table 3, Fig. 7)

Example 7

For the set of cervical scrapes used in Example 6 DNA was extracted and bisulphite-converted using the EZ DNA Methylation Kit (Zymo Research, Irvine, USA). For FAM19A4 methylation analysis was performed by quantitative methylation specific PCR (De Strooper et al., Int J Cancer. 2016 ;138(4):992-1002). All samples scored positive for FAM19A4 methylation at the threshold set at 70% specificity.

Interestingly, our 3-miRNA classifier was complementary to FAM19A4 methylation (Fig.8).

Example 8

Classifier analysis for self-samples

In the study described in Example 3 it was further assessed whether a combination of miRNAs would improve the detection of CIN3 and cancer.

To determine the optimal miRNA marker panel we used the data described in Example 2 and included extra hrHPV-positive self- samples samples totaling to 101 control women with either histologically confirmed <CIN1 or that displayed hrHPV clearance combined with normal cytology in follow-up (hereafter referred to as controls; median age of 42; range 33-63), 48 women who were histologically diagnosed with a CIN3 lesion (median age of 40; range 33-58) and 41 women who were histologically diagnosed with squamous cell carcinoma (SCC) (median age of 47; range 27-83)..

. We performed multivariable logistic regression followed by backward

elimination to identify the best combination of miRNA markers .In this analysis we combined CIN3 and cervical cancer (CIN3+) . The best clinical performance was achieved by multivariable logistic regression with a panel of 5 miRNAs (let- 7b, miR-15b, miR-20a, miR-93, and miR-222) for which we obtained an AUC of 0.78 (95% confidence interval (CI): 0.7173-0.8479) for CIN3+ detection (Figure 9A). Expectedly we observed a better clinical performance for the 5-miRNA marker panel compared to the individual clinical performance of the miRNAs (Figure 9B) At the threshold corresponding to 65% specificity in HPV-positive controls, the 5- miRNA marker panel revealed a sensitivity of 67% for CIN3 detection.

Importantly, 93% (38 out of 41) of the cervical cancers were detected.

Claims

Claims 1. A method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- measuring in a cell derived from a sample from a subject suspected having, or running a risk in developing, said lesions or carcinomas, the expression of the product of any of the microRNA (miR)-9, -15b,- 20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes;

- comparing said levels with the levels of healthy controls, wherein said sample is determined as positive if the normalised expression levels deviate from the control normalised expression levels.

2. The method according to claim 1, wherein the sample is obtained from cervical scraping or smears and wherein normalisation takes place on basis of the expression levels of miR-423 and one or both of RNU43 and RNU24.

3. The method according to claim 1, wherein the sample is obtained from self-collected cervico-vaginal specimens and wherein

normalisation takes place on basis of the expression levels of miR-423 and miR30b.

4. A method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising: a) measuring in a cell derived from a urine sample from a subject suspected having, or running a risk in developing, said lesions or carcinomas, the expression of the product of any of the microRNA (miE)-9, -21, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes, wherein said sample is determined as positive if the expression levels deviate from the control expression levels b) normalising said expression levels on basis of expression of miR- 423, or normalising said expression levels by taking the ratio of expression of two of the microRNAs in step a), preferably where the ratio of miR-21 over miR-9.

5. Method according to any of claims 1 - 4, wherein said HPV-induced high-grade precancerous lesion or HPV-induced invasive carcinoma is a high-grade premalignant cervical lesion or invasive cervical cancer.

6. Method according to any of claims 1 - 4, wherein said HPV-induced invasive cancer is a high-risk HPV-induced invasive cancer

7. Method according to any of the previous claims wherein the method involves nanotechnology, preferably lab on a chip technology.

8. Method according to any of the previous claims, where detection of the expression levels is performed by PCR, preferably reverse transcriptase PCR, more preferably quantitative rtPCR.

9. Use of the level of at least normalized expression of the product of any of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, - 203, -222, -375 and let-7b-5p genes as a molecular diagnostic marker for the detection of HPV-induced high-grade precancerous lesion or HPV-induced invasive carcinoma or nonHPV- induced gynaecological or anogenital cancer, preferably wherein the normalized gene product levels of said marker is predictive for the occurrence of said lesion, carcinoma or cancer.

10. Use of the reference gene miR-423 and one or more of miR30b,

RNU43 and RNU24 a for normalisation of expression levels of the product of any of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p in the detection of HPV- induced high-grade precancerous lesion or HPV-induced invasive carcinoma or nonHPV-induced gynaecological or anogenital cancer.

11. Use according to claim 10, where the reference genes miR-423 and miR30b are used in the detection in self-collected cervico-vaginal specimens.

12. Use according to claim 11, where the reference genes miR-423 and one or both of RNU43 and RNU24 are used in the detection in cervical scrapes.

13. Kit of parts for use in a method of detecting HPV-induced high- grade precancerous lesion or HPV-induced invasive carcinoma or nonHPV- induced gynaecological or anogenital cancer, said kit comprising means for the detection of HPV infection, wherein said means comprise probes and primers specific for hrHPV virus and wherein said kit further comprises means for the detection of expression of the product of any of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes and optionally means for the detection of expression of the product of the reference gene miR-423 and one or more of miR30b, RNU43 and RNU24.

14. A method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- measuring in a cell derived from a sample from a subject suspected having, or running a risk in developing said lesions or carcinomas, the expression of the product of any classifier comprised of two or more of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, - 149, -203, -222, -375 and let-7b-5p genes, preferably wherein said classifier is composed of the two miRNAs miR-15b and miR-375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let- 7b, miR-15b, miR-20a, miR-93, and miR-222;

- comparing said levels with the levels of healthy controls,

15. Kit of parts for use in a method of detecting HPV-induced high- grade precancerous lesion or HPV-induced invasive carcinoma or nonHPV- induced gynaecological or anogenital cancer, said kit comprising means for the detection of HPV infection, wherein said means comprise probes and primers specific for hrHPV virus and wherein said kit further comprises means for the detection of expression of any classifier comprised of two or more of the microRNA (miR)-9, - 15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes, preferably wherein said classifier is composed of the two miRNAs miR-15b and miR-375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let-7b, miR-15b, miR-20a, miR-93, and miR-222and optionally means for the detection of expression of the product of the reference gene miR-423 and one or more of miR30b, RNU43 and RNU24.

16. A method for detecting HPV-induced high-grade precancerous lesions and HPV-induced invasive carcinomas comprising:

- measuring in a cell derived from a sample from a subject suspected having, or running a risk in developing said lesions or carcinomas, the expression one or more of the microRNA (miR)-9, -15b, -20a,- 28, - 31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p or the expression of the product of any classifier comprised of two or more of the microRNA (miR)-9, -15b,-20a,- 28, -31, -93, -100,- 125b, -149, -203, -222, -375 and let-7b-5p genes preferably wherein said classifier is composed of the two miRNAs miR-15b and miR-375, or of the three miRNAs miR-149, miR-20a and miR-93, or of the five miRNAs let-7b, miR-15b, miR-20a, miR-93, and miR-222;

- comparing said levels with the levels of healthy controls,

wherein said sample is determined as positive if the normalised expression levels of the classifier and/or of the methylation marker deviate from the control normalised expression levels.