WO2019234176A1 - Novel biomarkers for carcinoma diagnosis - Google Patents

Abstract

The present invention relates a method for detecting high grade dysplasia, for assaying the potential for the development of carcinoma and for verifying whether a sample, which was determined to contain cells with high grad dysplasia, indeed contains cells with high grade dysplasia. Further provided is the use of agents for the detection of Sec62 and Ki67 in an in vitro-method for the detection of high grade dysplasia and for the reduction of false-positive results. Furthermore, a kit for the detection of high grad dysplasia is provided as well.

Description

NOVEL BIOMARKERS FOR CARCINOMA DIAGNOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims the benefit of priority of Luxemburg Patent Application No. 100824 filed 7 June 2018, the content of which is hereby incorporated by reference it its entirety for all purposes.

TECHNICAL FIELD OF THE INVENTION

[2] The present invention relates to a method for detecting high grade dysplasia, for assaying the potential for the development of carcinoma and for verifying whether a sample, which was determined to contain cells with high grad dysplasia, indeed contains cells with high grade dysplasia. Further provided is the use of agents for the detection of Sec62 and Ki67 in an in vitro- method for the detection of high grade dysplasia and for the reduction of false-positive results. Furthermore, a kit for the detection of high grad dysplasia is provided as well.

BACKGROUND

[3] Cervical cancer is the fourth most common cancer diagnosed and the fourth leading cause of cancer-related death with approximately 527,600 new cases and 265,700 deaths worldwide in 2012 (Ferlay et al., 2015). It was the second most common cancer among women in the 1970s, but mortality, thanks to cancer care, has fallen drastically in developed countries (Parkin, Stjernsward and Muir, 1984, Laara, Day and Hakama, 1987). In developing regions without cervical screening program, the incidence remains high.

[4] The Papanicolaou test (abbreviated as Pap test, also known as Pap smear, cervical smear, or smear test) is a method of cervical screening used to detect potentially pre-cancerous and cancerous processes in the cervix. Abnormal findings are often followed up by more sensitive diagnostic procedures, and, if warranted, interventions that aim to prevent progression to cervical cancer. The test was invented by, and named for, the Greek physician Georgios Papanicolaou.

[5] A systematic review by Nanda et al. found the sensitivity of Pap smear to be 51% (range 30% to 87%) and specificity to be 98% (range 86% to 100%) (Nanda et al., 2000). Since the sensitivity and specificity of cervical cytology varies widely numerous biomarkers for squamous epithelial lesions have been investigated and established (Nanda et al., 2000, Sano et al., 1998, Schmidt et al., 201 1 , Petry et al., 201 1 , Bergeron et al., 2015, Shroyer et al., 2006, Tosuner et al., 2017). [6] The detection of p16/KΪ-67 co-expression facilitates the identification of cells with deregulated cell cycle in cervical cytology samples, regardless of morphology-based interpretation parameters. The presence of 1 or more double immuno-reactive cells may be an indicator of an underlying CIN (cervical intraepithelial neoplasia) (Schmidt et al., 201 1 , Petry et al., 201 1 , Waldstrom et al., 2013, Bergeron et al., 2015. Schmidt et al. investigated in liquid- based cytology samples in parallel with p16/KΪ67 dual stain (Schmidt et al., 2011 ). The p16/KΪ67 dual stain was positive for CIN2 + in 92.2 % and for LSIL (low-grad squamous intraepithelial lesion) in 52.5 % of the cases and, in addition to similar sensitivity, had a higher specificity than detection of high-risk HPV (human papilloma virus) in LSIL and HSIL (Schmidt et al., 201 1 ).

[7] Despite increasing quality standards in cytology and the introduction of HPV test in screening programs, there is a persisting need for increasing the sensitivity and specificity in cancer screening, thereby also reducing the amount of unnecessary surgical procedures. The technical problem therefore is to comply with this need.

SUMMARY OF THE INVENTION

[8] The technical problem is solved by the subject-matter as defined in the claims. As shown in Example 1 , the combination of Sec62 and Ki67 is a reliable set of biomarkers for the detection of high grade dysplasia as it is negative for low grade dysplasia, such as CIN1 or CIN2. The same is shown in Example 2 for samples obtained from the vulva. Accordingly, provided herein is a method for detecting high grade dysplasia, for assaying the potential for the development of carcinoma, or for verifying whether a sample, which was determined to contain cells with high grad dysplasia, indeed contains cells with high grade dysplasia. These methods rely on the detection of Sec62 and Ki67, wherein the presence of Sec62 and Ki67 is indicative for high grade dysplasia. Further provided is the use of agents for the detection of Sec62 and Ki67 in an in vitro-method for the detection of high grade dysplasia and for the reduction of false-positive results. Furthermore, a kit for the detection of high grade dysplasia is provided as well.

[9] Accordingly, the present invention relates to a method for detecting high grade dysplasia in a sample obtained from a subject, said sample comprising cells, said method comprising (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample indicates that said sample comprises cells characterized by high grade dysplasia.

[10] Preferably, the high grade dysplasia is a CIN3 (cervical intraepithelial neoplasia grade 3) lesion, a CIN 3+ lesion, or carcinoma in situ (CIS).

[11] Preferably, the high grade dysplasia is a VIN3 (vulvar intraepithelial neoplasia grade 3), a VIN3+ lesion, or carcinoma in situ (CIS). [12] Preferably, the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, CIN2 lesion, CIN2+ lesion or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy).

[13] The present invention relates to a method for assaying the potential for the development of a carcinoma in a sample obtained from a subject, said sample comprising cells, said method comprising (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for a high potential for the development of a carcinoma, preferably a cervical or vulvar carcinoma.

[14] The present invention further relates to a method for verifying whether a sample, which was determined to contain cells with high grade dysplasia, obtained from a subject indeed contains cells with high grade dysplasia, said sample comprising cells, said method comprising: (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample indicates that the sample indeed contains cells with high grade dysplasia.

[15] Preferably, the sample is obtained from the urogenital tract, the anus or the otolaryngological regions.

[16] Preferably, the sample is obtained from the cervix or the vulva.

[17] Preferably, the sample is a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine.

[18] Preferably, the swab sample is a Pap smear sample.

[19] Preferably, the subject is human.

[20] Preferably, the human is a pregnant woman.

[21] Preferably, the subject is positive in a Pap smear test and/or positive for biomarkers p16 and Ki67.

[22] Preferably, the detection is performed by histology, DNA analysis, RNA analysis or protein analysis.

[23] Preferably, the detection is carried out by immuno-staining.

[24] Preferably, the presence of Sec62 and Ki67 in cells of said sample is indicative for the necessity of a biopsy and/or colposcopy, whereas the absence of Sec62 and Ki67 is indicative that routine testing should be performed.

[25] Preferably, the detection of Sec62 and Ki67 is performed in squamous cells only. [26] Preferably, the detection of Sec62 and Ki67 is not performed in reactive glandular cells.

[27] The present invention further relates to a use of agents for the specific detection of Sec62 and Ki67 in an in vitro method for the detection of high grade dysplasia in a sample obtained from a subject.

[28] The present invention further relates to a use of agents for the specific detection of Sec62 and Ki67 for the prevention of a false-positive result of a sample obtained from a subject, wherein the false-positive result is a false classification of said sample, which was determined to contain high grade dysplasia cells.

[29] The present invention also relates to a kit for detection high grade dysplasia in a sample comprising means for the specific detection of Sec62 and Ki67.

BRIEF DESCRIPTION OF THE DRAWINGS

[30] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, respectively. The Figures show:

[31] Fig. 1 : Immunocytochemical features of Sec62/Ki67-expression in cervical liquid-based cytology (LBC) specimens. The samples were stained for Sec62 (ER-located) and Ki67 (nucleus) and different intensities are shown from negative to CIN3.

[32] Fig. 2: Sec62/Ki67 dual stain (A, B), p16/KΪ67 dual stain (C, D), Sec62 immunostaining intensity grade (E) and SEC62 immunostaining intensity grade (F) according to cytological (A, C, E) and histological (B, D, F) findings. (NILM - negative for intraepithelial lesion or malignancy; AGC NOS - atypical glandular cells not otherwise specified; ASC-US - atypical squamous cells of undetermined significance; LSIL - low grade squamous intraepithelial lesion; ASC - H atypical squamous cells - cannot exclude HSIL; HSIL - high grade squamous intraepithelial lesion, HSIL inv. - high grade squamous intraepithelial lesion suspicious for invasion; SCC - squamous cell carcinoma).

[33] Fig. 3: Immunocytochemical features of Sec62/Ki67 expression in vulvar LBC specimens. (A) Cells negative for Sec62/Ki67 dual staining, as well as reactive squamous epithelial cells with high-intensity Sec62 staining without Ki67 positivity. Dual-stain-positive vulvar specimens: Sec62 immunostaining intensity grade 1 (B), 2 (C), 3 (D) and 4 (E). (F) Cases with VIN/SCC according to the Sec62 staining intensity. No normal/LSIL case showed Sec62/Ki67 positivity, and all SCC cases showed a high (grade 3 or 4) staining intensity.

[34] Fig. 4: p16/KΪ67 immunocytochemistry in vulvar cytology. (A) Negative and (B) positive for the p16/K67 dual stain. DETAILED DESCRIPTION OF THE INVENTION

[35] The present invention is described in detail in the following and is also illustrated by the appended examples and figures.

[36] The inventors surprisingly found that Sec62/Ki67 dual stain immunocytochemistry is useful for cytologists in the interpretation of high-grade squamous epithelium lesions on cytological specimens. Previous p16 single-stain immunocytochemistry protocols (see e.g. WO 00/01845) required the morphological interpretation of immune-reactive cells to distinguish between p16-positive cells exhibiting dysplasia and those cervical cells that occasionally overexpress p16 for physiological reasons such as squamous cell metaplasia cells or endocervical cells. The p16/KΪ67 dual stain (see e.g. WO 2004/038418) however tends to also detect low grade dysplasia. The Sec62/Ki67 dual stain, however, showed no false positivity in the squamous epithelium of the cervix (c.f. Example 1 , Figure 2 and Tables 1 and 2) or the vulva (c.f. Example 2, Table 6). Thus, the Sec62/Ki67 dual stain is especially suitable to detect high grade dysplasia and/or may also be used to verify a previous positive test result of another assay, e.g. those assays applied in the art. E.g., the p16/KΪ67 dual stain may also be positive for low grade dysplasias such as a CIN2 lesion. To differentiate between a CIN2 lesion, which possesses a high potential for self-healing and in many cases does not develop into a carcinoma, and a CIN3 lesion, which is already precancerous and dangerous, the Sec62/Ki67 dual stain of the present invention and as described herein could be applied as additional test.

[37] Sec62, also known as translocation protein Sec62, as used herein is a protein that is part of the protein translocation apparatus of the endoplasmatic reticulum membrane: the Sec complex comprised of Sec61 , Sec62 and Sec63. Sec62 as used herein encompasses Sec62 from eukaryotes, preferably mammals, more preferably from humans. A preferred Sec62 is shown in UniProt database entry Q99442 of 1 May 1997. A preferred Sec62 is shown in the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. Also encompassed by said Sec62 is a RNA transcript that has 50 %, 60 %, 70 %, 80 %, 90 % or 95 % or more identity over its entire nucleotide sequence with SEQ ID NO: 1. Also encompassed by said Sec62 is an expression product of an RNA transcript that has 50 %, 60 %, 70 %, 80 %, 90 % or 95 % or more identity over its entire amino acid sequence with in SEQ ID NO: 2. The percentage of sequence identity can, for example, be determined as described herein.

[38] KΪ67, also known as Antigen KI-67 or MKI67, as used herein is a nuclear protein that is associated with and may be necessary for cellular proliferation. Furthermore, it is associated with ribosomal RNA transcription. Ki67 as used herein encompasses Ki67 from eukaryotes, preferably mammals, more preferably from humans. A preferred Ki67 is shown in UniProt database entry P46013-1 , version 2 of 28 November 2016. A preferred Ki67 is shown in the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4. Also encompassed by said Ki67 is a RNA transcript that has 50 %, 60 %, 70 %, 80 %, 90 % or 95 % or more identity over its entire nucleotide sequence with SEQ ID NO: 3. Also encompassed by said Ki67 is an expression product of an RNA transcript that has 50 %, 60 %, 70 %, 80 %, 90 % or 95 % or more identity over its entire amino acid sequence with in SEQ ID NO: 4. The percentage of sequence identity can, for example, be determined as described herein.

[39] A variety of sequence based alignment methodologies, which are well known to those skilled in the art, can be used to determine identity among sequences. These include, but are not limited to, the local identity/homology algorithm of Smith, F. and Waterman, M. S. (1981 ) Adv. Appl. Math. 2: 482-89, homology alignment algorithm of Peason, W. R. and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85: 2444-48, Basic Local Alignment Search Tool (BLAST) described by Altschul, S. F. et al. (1990) J. Mol. Biol. 215: 403-10, or the Best Fit program described by Devereau, J. et al. (1984) Nucleic Acids. Res. 12: 387-95, and the FastA and TFASTA alignment programs, preferably using default settings or by inspection. Alternatively, an alignment may be done manually/visually for amino acids sequences as follows: the percent identity between an amino acid sequence in question (query sequence) and the amino acid sequence of Sec62 or Ki67 (reference sequence), respectively, as defined herein is determined by pairwise alignment in such a way that the maximum identity is obtained between both amino acid sequences. The identical amino acid residues between both amino acid sequences are counted and divided by the total number of residues of the amino acid sequence of Sec62 or Ki67 (including positions that do not contain amino acid residues, e.g. one or more gaps) yielding the percentage of identity. A preferred amino acid reference sequence of Sec62 is shown in SEQ ID NO: 2. A preferred amino acid reference sequence of Ki67 is shown in SEQ ID NO: 4.

[40] A similar method applies to nucleotide sequences: An alignment may be done manually/visually for nucleotide sequences as follows: the percent identity between an nucleotide sequence in question and the nucleotide sequence of Sec62 or Ki67 (reference sequence), respectively, as defined herein is determined by pairwise alignment in such a way that the maximum identity is obtained between both nucleotide sequences. The identical nucleotides between both nucleotide sequences are counted and divided by the total number of nucleotides of the nucleotide sequence of Sec62 or Ki67 (including positions that do not contain nucleotides, e.g. one or more gaps) yielding the percentage of identity. A preferred nucleotide reference sequence of Sec62 is shown in SEQ ID NO: 1. A preferred nucleotide reference sequence of Ki67 is shown in SEQ ID NO: 3.

[41] Accordingly, the present invention relates to a method for detecting high grade dysplasia in a sample obtained from a subject, said sample comprising cells, said method comprising (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample indicates that said sample comprises cells characterized by high grade dysplasia.

[42] Dysplasia in general, is a term used in pathology to refer to an abnormality of development or an epithelial anomaly of growth and differentiation (epithelial dysplasia). Cells or tissues showing dysplasia may develop cancer and as such, dysplasia is a precancerous condition. Dysplasia may be divided in“high grade” or“low grade” dysplasia. A“higher” grade of dysplasia is accompanied by increased abnormality and an increased risk of developing cancer. “High grade dysplasia” as used herein relates to a form of dysplasia, which most likely will result in a carcinoma.

[43] Dysplasia may however also relate to a precancerous lesion. The precancerous lesion can for example be a CIN (cervical intraepithelial neoplasia) lesion, an AIN (anal intraepithelial neoplasia) lesion, a VIN (vulvar intraepithelial neoplasia) lesion, a PIN (penile intraepithelial neoplasia) lesion, a VAIN (vaginal intraepithelial neoplasia) lesion, an urothelial neoplasia, or a SIN (oral and/or squamous intraepithelial neoplasia). The term “OIN” may also be used interchangeably when dysplasia as used herein relates to an oral intraepithelial neoplasia.

[44] Preferably, the dysplasia is a CIN. “Cervical intraepithelial neoplasia" or”CIN“ is a precancerous lesion that may exist at any one of three stages: CIN1 , CIN2, or CIN3. In CIN1 there is good maturation with minimal nuclear abnormalities and few mitotic figures. Undifferentiated cells are confined to the deeper layers (lower third) of the epithelium. Mitotic figures are present, but not very numerous. Cytopathic changes due to HPV infection may be observed in the full thickness of the epithelium. CIN2 is characterized by dysplastic cellular changes mostly restricted to the lower half or the lower two thirds of the epithelium, with more marked nuclear abnormalities than in Cl N 1. Mitotic figures may be seen throughout the lower half of the epithelium. In CIN3, differentiation and stratification may be totally absent or present only in the superficial quarter of the epithelium with numerous mitotic figures. Nuclear abnormalities extend throughout the thickness of the epithelium. Many mitotic figures have abnormal forms.“High grade dysplasia” within the context of the invention may relate to a CIN2, CIN2+, CIN3, CIN3+ lesion or even carcinoma in situ (CIS). Preferably,“high grade dysplasia” is a CIN3 lesion, a CIN3+ lesion or CIS. High grade dysplasia may also be a carcinoma in situ. High grade dysplasia can also be a CIN2+ lesion. A CIN2+ lesion includes CIN2 and CIN3 lesions. Methods for detecting and grading CIN lesions are known to the skilled artesian and for example described in Barron et al. (2014)“Low-grade squamous intraepithelial lesion/cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) is a unique category of cytological abnormality associated with distinctive HPV and histopathologic CIN 2+ detection rates.” Am J Clin Pathol;141 (2):239-46. [45] The precancerous lesion can also be a low grade or a high grade precancerous lesion. The risk of low grade precancerous lesion for transforming into high grade HPV-induced precancerous lesion, and eventually cancer, is low and has e.g. been described by Bansal et al. (2008) Natural History of Established Low Grade Cervical Intraepithelial (CIN 1 ) Lesions. Anticancer Research 28: 1763-1766. This means that a subject with low grade dysplasia can have a risk of developing cancer of not more than 50 %, 40 % 30 %, 20 %, 10 % 5 %, 3 %, 1 %, 0 %.

[46] The low grade precancerous lesions may also be a low grade squamous intraepithelial lesion (LSIL). The LSIL is well known to the skilled artesian and has been termed by the Bethesda System terminology. For example, LSIL is described in Mukhopadhyay et al. (2013) “Evaluation of the category high-grade squamous intraepithelial lesion in The Bethesda System for reporting cervical cytology.” J Cytol. Jan-Mar; 30(1 ): 33-35. The LSIL may be a CIN1 or CIN2 lesion, wherein the CIN2 lesion can be negative for Sec62 and Ki67 as described herein.

[47] High grade dysplasia represents a more advanced progression towards malignant transformation. The risk of high grade dysplasia for transforming to cancer is high. This means that a subject with a high grade dysplasia may have a risk of developing cancer of more than 20 %, 30 %, 40 % 50 %, 60 % 70 %, 80 %, 90 %, 95 %, 93 %, 97 % 99 % or 100 %. High grade dysplasia may be caused by transforming infections or an HPV infection.

[48] A high grade dysplasia can for example be a high-grade squamous intraepithelial lesion (HSIL). High grade squamous intraepithelial lesions are well known to the skilled artesian and have also been termed by the Bethesda System terminology and are e.g. described in Mukhopadhyay et al. (2013)“Evaluation of the category high-grade squamous intraepithelial lesion in The Bethesda System for reporting cervical cytology.” J Cytol. Jan-Mar; 30(1 ): 33-35. These lesions include moderate dysplasia (CIN2), severe dysplasia (CIN3), and carcinoma in situ (CIS). The high grade squamous intraepithelial lesion may be a CIN3 lesion.

[49] In addition or alternatively, the lesion can be a carcinoma in situ (CIS) such as for example an adenocarcinoma in situ, a squamous cell carcinoma in situ, a small cell carcinoma in situ, a neuroendocrine tumor in situ, a glassy cell carcinoma in situ or a villoglandular adenocarcinoma in situ. The Dysplasia can also be an adenocarcinoma in situ or a squamous cell carcinoma in situ.

[50] The classification of precancerous lesions (CIN) can also be used for classification of intraepithelial lesions in other regions of the body. In some regions of the body, however, a different terminology can be used. [51] For example, the dysplasia can also be a conjunctival intraepithelial neoplasia (CIN). These are known to the skilled artesian and for example described in Huerva and Ascaso Conjunctival Intraepithelial Neoplasia -Clinical Presentation, Diagnosis and Treatment Possibilities published in Intraepithelial Neoplasia edited by Dr. Supriya Srivastava; Publisher InTech Published online 08, February, 2012. Clinically, CIN can appear as a fleshy, sessile or minimally elevated lesion at limbus in the interpalpebral fissure or in the forniceal or tarsal conjunctiva. The limbal lesion may extend for a variable distance into the epithelium of the adjacent cornea. A white plaque (leukoplakia) may occur on the surface of the lesion due to secondary hyperkeratosis.

[52] When the abnormal conjunctival epithelial cellular proliferation involves only partially the epithelium thickness it can be classified as mild or low grade CIN, a condition also called mild or moderate dysplasia. When it affects full thickness epithelium it can be called severe or high grade CIN, a condition also called severe dysplasia. In these cases there may be an intact surface layer of cells. Where there are no longer normal surface cells then the process is termed carcinoma in situ. Histopathologically, mild CIN (dysplasia) is characterized by a partial thickness replacement of the surface epithelium by abnormal epithelial cells which lack of normal maturation. Severe CIN (severe dysplasia) is characterized by a nearly full-thickness replacement of the epithelium by similar cells. Accordingly, the high grade dysplasia may be a high grade or severe conjunctival intraepithelial neoplasia.

[53] The Dysplasia can also be an anal squamous intraepithelial lesion (ASIL), sometimes also referred to as AIN. These lesions are known to the skilled person and for example described in Pineda and Welton (2009) “Management of Anal Squamous Intraepithelial Lesions.” Clin Colon Rectal Surg. 22(2): 94-101 and Hoots, Palefsky, Pimenta and Smith (2009)“Human papillomavirus type distribution in anal cancer and anal intraepithelial lesions” Int. J. Cancer: 124, 2375-2383. ASIL can be classified into two groups based on the degree of dysplasia: low-grade SIL (LSIL or AIN1 ), which is not a direct precursor of cancer but may lead to high-grade SIL (HSIL) later on, and high-grade SIL (HSIL), which can be precursor of a carcinoma in situ and/or invasive anal cancer. Accordingly, the high grade dysplasia may be a high grade ASIL or HSIL.

[54] LSILs demonstrate nuclear atypia and perinuclear cytoplasmic cavitation, with a nucleus that is larger than that of a normal intermediate squamous cell on cytology. These lesions are characterized by low nuclear/cytoplasmic ratios (koilocytes), atypical cells confined to superficial layers, and mitotic activity in the lower third of the CIN epithelium.

[55] HSILs demonstrate high nuclear/cytoplasmic ratios on cytology, with cell sizes smaller than those with LSIL. There are high nuclear/cytoplasmic ratios and full-thickness atypia, which includes parabasal atypia, loss of cell polarity, and mitotic activity in the upper third of the mucosa, as well as abnormal mitotic figures. Full-thickness atypia with invasion of the basement membrane defines anal squamous cell carcinoma (SCC). The definition HSIL has been accepted by the American Joint Committee on Cancer (AJCC) and is used in the seventh edition of their staging manual. Thus, the high grade dysplasia can be a HSIL.

[56] The dysplasia preferably is a squamous vulvar intraepithelial neoplasia (VIN). Also VIN are known to the person skilled in the art and for example described in Sideri et al. (2005) “Squamous Vulvar Intraepithelial Neoplasia.” J. Reprod Med; 50:807-810.

[57] Traditionally, pathologists classified VIN into 3 grades: VIN 1 , 2 or 3, in keeping with cervical intraepithelial neoplasia (CIN 1 , 2, 3). Notably, VIN3 describes a carcinoma in situ. In 2004, the International Society for the Study of Vulvovaginal Diseases (ISSVD) reclassified VIN. They recommended that the term VIN 1 , previously used to describe a mild change in the lower epithelial lining, should no longer be used, as these changes have been found to be due to irritation or non-precancerous viral wart infection and often clear up without treatment. The ISSVD recommended that the term VIN should be used for high-grade abnormal squamous lesions (these were previously known as VIN 2 and VIN 3). VIN (usual-type or undifferentiated type) can be described by the pathologist as warty, basaloid or mixed VIN. These types of VIN are due to infection with cancer-forming (oncogenic) types of human papillomavirus (HPV). It can be solitary or multicentric. So, VIN2 and VIN3 can be considered as high grade dysplasia.

[58] The Dysplasia can also be penile intraepithelial neoplasia (PeIN) sometimes also referred to as PIN. This neoplasia is a premalignant lesion that can affect the glans, prepuce, or the shaft of the penis. On the basis of the degree of dysplasia, PeIN is divided into PeIN 1 (mild), PeIN 2 (moderate), and PeIN 3 (severe). PelN2 and 3 can be precancerous lesions. PelN3 is also called carcinoma in situ. Accordingly, the high grade dysplasia may also be a PeIN 2 or PeIN 3 lesion.

[59] The precancerous lesion can also be a vaginal intraepithelial neoplasia (VAIN). This lesions are known to the skilled artesian and for example described in Velazquez, Chaux, Cubilla Histologic classification of penile intraepithelial neoplasia. Semin Diagn Pathol. 2012 May;29(2):96-102. VAIN describe a condition that describes premalignant histological findings in the vagina characterized by dysplastic changes. VAIN can be detected by the presence of abnormal cells in a Papanicolaou test (Pap smear). Like cervical intraepithelial neoplasia, VAIN comes in three stages, VAIN1 , 2, and 3. In VAIN1 , a third of the thickness of the cells in the vaginal skin is abnormal, while in VAIN3, the full thickness is affected. VAIN3 is also known as carcinoma in situ. Thus, the high grade dysplasia can be a VAIN2 or VAIN 3 lesion. The high grade dysplasia can be a VAIN3 lesion. [60] The precancerous lesion can also be an oral epithelial dysplasia or squamous intraepithelial neoplasia (SIN). These lesions are known to the skilled artesian and for example described in Speight Update on Oral Epithelial Dysplasia and Progression to Cancer. Head Neck Pathol. 2007 Sep; 1 (1 ): 61-66.

[61] Mild dysplasia (grade l/SIN 1 ) demonstrates proliferation or hyperplasia of cells of the basal and parabasal layers which does not extend beyond the lower third of the epithelium. Cytological atypia is generally slight with only mild pleomorphism of cells or nuclei. Mitoses are not prominent, and when present are usually basally located and normal. Architectural changes are minimal. Moderate dysplasia (grade II/SIN2) demonstrates a proliferation of atypical cells extending into the middle one-third of the epithelium. The cytological changes are more severe than in mild dysplasia and changes such as hyperchromatism, and prominent cell and nuclear pleomorphism may be seen. Increased and abnormal mitoses may be present, but these are usually located in the basal layers. Architectural changes may be seen in the lower half of the epithelium where there may be loss of basal polarity and hyperplasia leading to bulbous rete pegs. However stratification and maturation are relatively normal, often with hyperkeratosis. In severe dysplasia (grade III/SIN3) there is abnormal proliferation from the basal layer into the upper third of the epithelium. Cytological and architectural changes can be very prominent. All the changes seen in mild and moderate dysplasia are seen but in addition there is marked pleomorphism often with abnormally large nuclei with prominent or even multiple nucleoli. Prominent and suprabasal mitoses are usually evident and abnormal tripolar or star-shaped forms may be seen. Apoptotic bodies may also be prominent. Architectural changes are severe, often with complete loss of stratification and with deep abnormal keratinisation and even formation of keratin pearls. Although the epithelium may be thickened, severe dysplasia is sometimes accompanied by marked epithelial atrophy. This is especially prominent in lesions from the floor of mouth, ventral tongue or soft palate and may be a feature of lesions which have presented clinically as erythroplakia. In these cases there may be minimal evidence of stratification or keratinisation, and atypical cells may extend to the surface. Carcinoma in situ is the most severe form of epithelial dysplasia and is characterized by full thickness cytological and architectural changes. In the oral cavity such changes are rare, and often, even in the presence of the most severe atypia, there is still an intact keratinized surface layer. Thus, the high grade dysplasia can be a SIN2 or SIN3 lesion. The high grade dysplasia can also be a SIN3 lesion.

[62] Histological changes of the squamous epithelium that occur in the process of oral, oro- and hypopharyngeal and laryngeal carcinogenesis, can also cumulatively designated squamous intraepithelial lesions (SILs). The term SILs has been proposed as an all-embracing expression of the whole spectrum of epithelial changes ranging from squamous cell hyperplasia to carcinoma in situ. [63] Considering the high grade dysplasias or lesions as described herein, the high grade dysplasia can for example be selected from the group consisting of high-grade squamous epithelial lesions, CIN2+ or CIN3 (cervix uteri), severe CIN (cornea/conjunctiva), high grade SIL (ASIL; anorectal region/anus), VIN2/3 (vulva) or, SIN2 or SIN3 (oropharynx). The Dysplasia can also be selected from the group consisting of high-grade squamous epithelial lesions, CIN2+ or CIN3 (cervix uteri), severe CIN (cornea/conjunctiva), high grade SIL (ASIL; anorectal region/anus), VIN2/3 (vulva) or SIN2 or SIN3 (oropharynx), VAIN2/3 (vagina) or PelN2/3 (penis).

[64] In general, the high grade dysplasia can be selected from the group consisting of dysplasias of the cervix uteri, anus, vagina, vulva, penis, oropharynx such as tongue or tonsillar cancer, oropharynx, cornea/conjunctiva or skin. The high grade dysplasia can also be selected from skin, head and neck (oropharynx, nasopharynx), lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder. The high grade dysplasia can also be selected from cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva.

[65] As shown in Example 1 , the dual stain of Sec62 and Ki67 can be used to differentiate between high grad dysplasia and low grade dysplasia of the cervix and as shown in Example 2 the dual stain of Sec62 and Ki67 can be used to differentiate between high grade dysplasia and low grade dysplasia of the vulva. In this context, high grade dysplasia preferably relates to a CIN3 lesion, a CIN3+ lesion or carcinoma in situ. As described herein, the terminology using “intraepithelial neoplasia” (IN) is also used to describe the malignancy in other tissues of the anogenital tract. E.g., when describing a high grade dysplasia in vulva, the present invention relates to VIN3, VIN3+ or CIS. In one embodiment, high grade dysplasia may relate to CIN2+, CIN3, CIN3+ lesions and CIS. In another embodiment, high grade dysplasia may relate to CIN2, CIN2+, CIN3, CIN3+ lesions and CIS.

[66] In contrast, if one or both of Sec62 and Ki67 are absent in a sample, the lesion may be qualified as low-grade squamous cell intraepithelial lesion (LSI L) as defined by the Bethesda system. Accordingly, in one embodiment, the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, CIN2 lesion, CIN2+ lesion or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy). In another embodiment, the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, CIN2 lesion, or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy). In a further embodiment, the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy). [67] High grade dysplasia is associated with an increased risk for the development of a carcinoma. Hence, a positive dual staining of Sec62 and Ki67 may be also indicative for a risk of developing cancer. Accordingly, the invention further relates to a method for assaying the potential for the development of a carcinoma in a sample obtained from a subject, said sample comprising cells, said method comprising (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for a high potential for the development of a carcinoma.

[68] The term“potential for the development” when used in the context of development of a carcinoma describes the probability of the development of a carcinoma. The potential does not necessarily have to be 100 % accurate. This is so because - self-explanatory as it is - the methods of the invention cannot provide a 100 % safe prediction whether or not a patient may develop a carcinoma, since, apart from the expression of Sec62 and Ki67, individual factors such as age, body weight, general health, sex, diet, lifestyle, drug interaction and the like may have an influence on the probability of the development of a carcinoma. However, if a subject shows in a sample obtained from said subject expression of both Sec62 and Ki67, the potential, i.e. the likelihood or probability that the subject will develop a carcinoma is more than 50 %. Preferably, the likelihood or probability is more than 60%, 70%, 80% or 90%, more preferably more than 95%.

[69] The methods of the invention are in principle applicable to any tissue sample of an animal. Especially preferred are tissues of the epithelium, more preferably tissues comprising squamous (epithelial) cells. Squamous cells have the appearance of thin, flat plates that can look polygonal when viewed from above. The cells fit closely together in tissues; providing a smooth, low-friction surface over which fluids can move easily. The shape of the nucleus usually corresponds to the cell form and helps to identify the type of epithelium. Squamous cells tend to have horizontally flattened, nearly oval shaped nuclei because of the thin flattened form of the cell. Squamous epithelium is found lining surfaces such as the skin, and alveoli in the lung, enabling simple passive diffusion as also found in the alveolar epithelium in the lungs. Specialized squamous epithelium also forms the lining of cavities such as in blood vessels, as endothelium and in the pericardium, as mesothelium and in other body cavities. Especially preferred tissues, from which the sample can be obtained, are the urogenital tract, the anus or the otolaryngological regions. Accordingly, the sample is preferably obtained from the urogenital tract, the anus or the otolaryngological regions.

[70] It is further envisioned by the present invention that the sample has been obtained from a subject. Exemplary sites from which a sample can be obtained include cervix uteri, anorectal region, vagina, vulva, penis, oropharynx such as tongue or tonsil, conjunctiva/limbus or skin. The sample can also be obtained from the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. The sample can also be obtained from the cervix uteri or anorectal region. The sample may also be obtained from the cervix or the vulva.

[71] An exemplary swab sample can be a Pap (Papanicolaou) smear sample. The skilled person knows this type of test/sample, which is e.g. described in Biran G, Levy T. (2004) “Papanicolau smears: reducing the false negative rate by improving the method.” Harefuah;143(3):217-21 , 245. The Pap smear is commonly issued as a screening test for cervical cancer or cervical precancerous lesions. Cells scraped from the opening of the cervix are examined under a microscope. A Pap smear sample may however, also be obtained from the cervix uteri, anorectal region, vagina, vulva, penis, oropharynx such as tongue or tonsil, conjunctiva/limbus or skin. In particular a sample, such as a Pap smear sample can be obtained from the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. Alternatively, the sample such as the Pap smear sample can be obtained from the cervix or the vulva.

[72] Further, the tissue sample can for example be a conisation sample. Cervical conisation refers to an excision of a cone-shaped sample of tissue from the mucous membrane of the cervix. The biopsy/tissue sample that has been excised from the cervix can then be analyzed by the methods of the present invention. It is also possible to obtain a sample of a woman who has undergone conisation, to additionally analyze the sample for the presence of Sec62 and/or Ki67. For that e.g. a Pap smear sample may be used.

[73] In certain special embodiments of the present invention, the sample may be prepared as a monolayer or thin layer preparation of a cytological specimen. The respective methods for preparation of monolayer or thin-layer preparation in cytology are known to those of skill in the art. In one embodiment the preparation may e.g. comprise the ThinPrep technology. Other methods comprise conventional smears, or method employing suspensions of cells for preparation of the cytological specimens. Preparation of a sample may comprise e.g. obtaining a sample of a tissue, of a body fluid, of cells from a patient. According to the present invention, preparation of the sample may also comprise several steps of further preparations of the sample, such as preparation of dissections, preparation of cell suspensions, spreading or applying the cells to be examined onto microscopic slides, preparation of tissue arrays, isolation of polypeptides or nucleic acids, preparation of solid phase fixed peptides or nucleic acids or preparation of beads, membranes or slides to which the molecules to be determined are coupled covalently or non-covalently. In certain embodiments of the present invention the method may be performed in an automated manner. The automation of the method may be achieved by automated staining and analysis of histological or cytological specimens on a solid surface by microscopic means. In another embodiment the automation may comprise a flow- cytometric analysis of the staining of cells in solution.

[74] The subject may be a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Preferably, a mammal is a human, dog, cat, cow, pig, mouse, rat etc. The mammal can also be a human being. Thus, the subject can be a vertebrate, preferably a human. For a sample obtained from the cervix, vulva or uterus, the subject is a female and for a sample obtained from the penis, the subject is a male. The human may be between 21 years and 65 years, between 12 and 18 years, between 14 and 21 years, at least 10 years, at least 12 years, at least 14 years, at least 16 years, at least 18 years or at least 21 years. The human female may be between 21 years and 65 years, between 12 and 18 years, between 14 and 21 years, at least 10 years, at least 12 years, at least 14 years, at least 16 years, at least 18 years or at least 21 years.

[75] In one embodiment, the subject is a pregnant woman. As described herein, the present invention is suitable to detect high-grade dysplasia and therefore can be used to avoid unnecessary treatments or biopsies. This is of utmost importance for pregnant women, where a biopsy or further unnecessary procedures should be avoided to prevent damage from the mother or the embryo/fetus, i.e. to prevent premature birth or miscarriage.

[76] A person skilled in the art is capable of detecting specifically Sec62 and Ki67 or any other biomarker necessary. Standard methods for detection include, but are not limited to, histology, DNA analysis, RNA analysis, or protein analysis. These methods include methods such as PCR, qPCR, sequencing, SDS-PAGE, Western Blot, Northern Blot, Southern Blot and many more. Preferably, the detection is performed by histology. More preferably, the detection is carried out by immuno-staining, immunohistochemistry and/or immunocytology. Exemplary methods for the detection are shown in Examples 1 and 2. The application of these methods is known to a person skilled in the art.

[77] Immunohistochemical samples such as sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the tissue can be analyzed with immunohistochemistry. For immunocytology extracellular matrix and other stromal components can be removed, leaving only whole cells to stain. Therefore, immunocytology can include the analysis of cells obtained in a sample such as a smear sample. Both, immunohistochemistry and immunocytology can comprise the use of antibodies to detect the Sec62 and Ki67 protein. Thus, both immunohistochemistry and immunocytology can also comprise fluorescent or non-fluorescent immunohistochemistry and/or immunocytology.

[78] Exemplary means to specifically detect Sec62 and Ki67 protein that are suitable in SDS- PAGE, Western Blot, immuno-staining, immunohistochemistry and/or immunocytology can include suitable binding proteins directed e.g. against one of these molecules. The binding protein can be selected from the group consisting of an antibody, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule with antibody-like binding properties.

[79] Such an“antibody” can be a full length antibody, a recombinant antibody molecule, or a fully human antibody molecule. A full length antibody is any naturally occurring antibody. The term "antibody" also includes immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as lgG1 , lgG2 etc.). Such full length antibodies can be isolated from different animals such as e.g. different mammalian species. A "recombinant antibody molecule" refers to an antibody molecule the genes of which has been cloned, and is produced recombinantly in a host cell or organism, using well-known methodologies of genetic engineering. Typically, a recombinant antibody molecule has been genetically altered to comprise an amino acid sequence, which is not found in nature. Thus, a recombinant antibody molecule can be a chimeric antibody molecule or a humanized antibody molecule.

[80] The binding protein can also be an “antibody fragment”. Such antibody fragments comprise any part of an antibody, which comprises a binding site. Illustrative examples of such an antibody fragment are single chain variable fragments (scFv), Fv fragments, single domain antibodies, such as e.g. VHH (camelid) antibodies, di-scFvs, fragment antigen binding regions (Fab), F(ab')₂ fragments, Fab’ fragments, diabodies or domain antibodies, to name only a few (Holt et al (2003)“Domain antibodies: proteins for therapy.” Trends Biotechnol. 2003 Nov; 21 (1 1 ):484-90).

[81] The binding protein may also only have a single binding site, i.e., may be monovalent. Examples of monovalent binding proteins include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties. Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, a single-chain Fv fragment (scFv) or an scFv-Fc fragment. Furthermore, the antibody or antibody fragment may be monoclonal or polyclonal.

[82] The binding protein that can be used in this invention can be a monoclonal antibody or antibody fragment. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kohler and Milstein Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., US Patent 4,946,778) can be adapted to produce single chain antibodies to Sec62 or Ki67 polypeptides.

[83] The binding protein can also be a proteinaceous binding molecule with antibody-like binding properties. Exemplary but non-limiting proteinaceous binding molecules include an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, an avimer or a (recombinant) receptor protein.

[84] The diagnosis or presence of high grade dysplasia may be an indication to perform a biopsy and/or colposcopy. Accordingly, the presence of Sec62 and Ki67 in cells of said sample may be indicative for the necessity of a biopsy and/or colposcopy, whereas the absence of Sec62 and Ki67 is indicative that routine testing should be performed.“Biopsy” as used herein relates to a medical test involving extraction of sample cells or tissues for examination to determine the presence or extent of a disease.“Colposcopy” is a medical diagnostic procedure to examine an illuminated, magnified view of the cervix and the tissues of the vagina and vulva. Many premalignant lesions and malignant lesions in these areas have discernible characteristics which can be detected through the examination. It is done using a colposcope, which provides an enlarged view of the areas, allowing the colposcopist to visually distinguish normal from abnormal appearing tissue and take directed biopsies for further pathological examination. The main goal of colposcopy is to prevent cervical cancer by detecting precancerous lesions early and treating them. Complications of a colposcopy may include bleeding and/or infection at the biopsy site and/or endometrium but also miscarriage or premature birth in pregnant women.

[85] “ Routine testing” as used herein relates to standard procedures and test that are performed on a subject in the absence of a suspicion of a disease, i.e. normal screening procedure without any extraction of biological material. Routine testing may include Pap smear tests, tests for HPV infections or analysis of the presence of p16 or p16 and Ki67. This routine testing is usually performed at intervals between 1 and 3 years. Huh et al. 2015, J Lower Gen Tract Dis, 19:91-96, incorporated by reference in its entirety, published a protocol for routine testing that can also be applied in this context.

[86] Sec62/Ki67 can be positive in glandular cells. Therefore, the interpretation of the results may require that the evaluation of the staining is done only on the squamous cells and the nonspecific glandular cells should be ignored. However, it is to be understood that the presence of non-squamous cells in a sample does not impair the reliance of the present invention. Accordingly, the detection of Sec62 and Ki67 preferably should be performed in squamous cells only. In many tissues that comprise squamous cells also glandular cells may be found. Accordingly, the detection of Sec62 and Ki67 preferably is not performed in reactive glandular cells. “Reactive glandular cells” may be characterized as atypical glandular cells (AGC) endocervical, not otherwise specified (NOS), and/or AGC endocervical, favor neoplastic (AGC- FN) according to the Bethesda system, which is e.g. defined in Solomon D, Davey D, Kurman R et al.: The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 287 (2002) 2114-21 19.

[87] A false-positive identification of a sample as showing high-grade dysplasia may lead to unnecessary biopsies, operations etc. As shown in Table 2, the dual stain of p16 and Ki67 is positive for CIN2 lesions, while the combination of KI67 and Sec62 of the present invention does not falsely classify samples as positive, i.e. as high-grade dysplasia (see Table 1 ). Hence, in a situation, in which a subject is confronted with a positive result in another test, e.g. a dual stain of p16 and Ki67, the present invention may be used to verify the result. Accordingly, the present invention relates to a method for verifying whether a sample, which was determined to contain cells with high grade dysplasia, obtained from a subject indeed contains cells with high grade dysplasia, said sample comprising cells, said method comprising: (i) detecting Sec62 and Ki67 in cells of said sample, wherein the presence of Sec62 and Ki67 in cells of said sample indicates that the sample indeed contains cells with high grade dysplasia. In this context, the present invention also relates to the use of agents for the detection of Sec62 and Ki67 for the prevention of a false-positive result of a sample obtained from a subject, wherein the false- positive result is a false classification of said sample, which was determined to contain high grade dysplastic cells. Examples for other assays include the single stain of p16, dual stain p16/KΪ67, an HPV test or a Pap smear test.

[88] The present invention further relates to a kit for detection of high grade dysplasia in a sample comprising means for the specific detection of Sec62 and Ki67. The detection of Sec62 and/or Ki67 can be performed with any mean that is suitable for that purpose.“Specific” in this context describes a“mean” that only binds and/or detects Sec62, Ki67, or Sec62 and Ki67. Such means are also known to the skilled artesian and some of them are also described herein. For example, the mean for the specific detection of Sec62 and/or Ki67 can be a binding protein as described herein. Examples of such a binding protein can include an anti-Sec62 antibody and/or an anti-Ki67 antibody. The antibody can be a monoclonal or a polyclonal antibody. Preferably, the antibody can be a monoclonal antibody. The mean can also be a probe specific for Sec62 and/or Ki67. Means for the specific detection of Sec62 and Ki67 may comprise primer enabling the amplification of RNA transcripts of the biomarker and optionally one or more reference genes, and optionally at least one of means for RNA extraction, for transcription of RNA to DNA and/or performing a PCR or qPCR reaction. [89] In this context, the present invention also relates to the use of agents for the detection of Sec62 and Ki67 in an in vitro method for the detection of high grade dysplasia in a sample obtained from a subject.“Agents for the detection of Sec62 and Ki67” as used herein relate to agents that are capable of specifically detecting Sec62 and/or Ki67 protein or RNA transcripts. The specific detection of Sec62 and/or Ki67 can be performed with any mean that is suitable for that purpose. Such means are also known to the skilled artesian and some of them are also described herein.“Specific” in this context describes a mean that only binds and/or detects Sec62, Ki67, or Sec62 and Ki67. For example, the mean for the specific detection of Sec62 and/or Ki67 can be a binding protein as described herein. Examples of such a binding protein can include an anti-Sec62 antibody and/or an anti-Ki67 antibody. The antibody can be a monoclonal or a polyclonal antibody. Preferably, the antibody can be a monoclonal antibody. The mean can also be a probe specific for Sec62 and/or Ki67. Means for the specific detection of Sec62 and Ki67 may comprise primer enabling the amplification of RNA transcripts of the biomarker and optionally one or more reference genes, and optionally at least one of means for RNA extraction, for transcription of RNA to DNA and/or performing a PCR or qPCR reaction.

[90] The present invention further relates to the following items:

1. A method for detecting high grade dysplasia in a sample obtained from a subject, said sample comprising cells, said method comprising

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample indicates that said sample comprises cells characterized by high grade dysplasia.

2. The method of item 1 , wherein the high grade dysplasia is a CIN3 (cervical intraepithelial neoplasia grade 3) lesion, a CIN 3+ lesion, or carcinoma in situ (CIS).

3. The method of item 1 or 2, wherein the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, CIN2 lesion, CIN2+ lesion or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy).

4. A method for assaying the potential for the development of a carcinoma in a sample obtained from a subject, said sample comprising cells, said method comprising

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for a high potential for the development of a cervical carcinoma.

5. The method of any one of items 1-4, wherein the sample is obtained from the urogenital tract, the anus or the otolaryngological regions.

6. The method of any one of items 1-5, wherein the sample is obtained from the cervix or the vulva.

7. The method of any one of items 1-6, wherein the sample is a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine. 8. The method of item 7, wherein the swab sample is a Pap smear sample.

9. The method of any one of items 1-8, wherein the subject is human.

10. The method of item 9, wherein the human is a pregnant woman.

1 1. The method of item 9 or 10, wherein the subject is positive in a Pap smear test and/or positive for biomarkers p16 and Ki67.

12. The method of any one of items 1-1 1 , wherein the detection is performed by histology, DNA analysis, RNA analysis or protein analysis.

13. The method of any one of items 1-1 1 , wherein the detection is carried out by immuno- staining.

14. The method of any one of items 1-13, wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for the necessity of a biopsy and/or colposcopy, whereas the absence of Sec62 and Ki67 is indicative that routine testing should be performed.

15. The method of any one of items 1-14, wherein the detection of Sec62 and Ki67 is performed in squamous cells only.

16. The method of any one of items 1-15, wherein the detection of Sec62 and Ki67 is not performed in reactive glandular cells.

17. A method for verifying whether a sample, which was determined to contain cells with high grade dysplasia, obtained from a subject indeed contains cells with high grade dysplasia, said sample comprising cells, said method comprising:

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample indicates that the sample indeed contains cells with high grade dysplasia.

18. Use of agents for the detection of Sec62 and Ki67 in an in vitro method for the detection of high grade dysplasia in a sample obtained from a subject.

19. Use of agents for the detection of Sec62 and Ki67 for the prevention of a false-positive result of a sample obtained from a subject, wherein the false-positive result is a false classification of said sample, which was determined to contain high grade dysplasia cells.

20. A kit for detection high grade dysplasia in a sample comprising means for the detection of Sec62 and Ki67.

[91] It is noted that as used herein, the singular forms“a”,“an”, and“the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to“a reagent” includes one or more of such different reagents and reference to“the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. [92] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[93] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[94] The term“less than” or in turn“more than” does not include the concrete number.

[95] For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, e.g. more than 80 % means more than or greater than the indicated number of 80 %.

[96] Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term“containing” or“including” or sometimes when used herein with the term“having”. When used herein“consisting of" excludes any element, step, or ingredient not specified.

[97] The term“including” means“including but not limited to”.“Including” and“including but not limited to” are used interchangeably.

[98] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[99] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[100] The content of all documents and patent documents cited herein is incorporated by reference in their entirety. EXAMPLES

[101] An even better understanding of the present invention and of its advantages will be evident from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

Example 1 : Dual staining of Sec62 and Ki-67 in cervical samples

Material and methods

Patients and tissue samples

[102] Residual cytological material from 100 ThinPrep (Hologic, Marlborough, Massachusetts) Papanicolaou (Pap) test liquid-based cytology (LBC) from patients who underwent colposcopy was available. Except for two NILM (negative for intraepithelial lesion or malignancy) cases histology was performed (63 excision, 35 target biopsies). p16/Ki67 dual stain results were known for all cases.

[103] All patients gave written consent for the use of their tissue samples according to the Declaration of Helsinki, and the study protocol was approved by the approval of local ethic board.

Sec62/Ki67 dual stain

[104] Sec62 and Ki67 immunostains were individually tested in different antibody dilutions (1 :400, 1 :600, 1 :800). The Sec62 staining in 1 :400 and the Ki67 staining in 1 :600 dilution proved to be successful. This was followed by a sequential dual stain procedure in different orders (Sec62 followed by Ki67 and Ki67 followed by Sec62) and with different dilutions (1 :400, 1 :600, 1 :800) in all possible combinations. Finally, simultaneous staining was tested with antibody cocktails with different dilutions of each antibody. In addition, individual steps of staining (fixation, unmasking, blocking) were performed with different methods to achieve the best possible results.

[105] Slides were prepared with the T2000 processor (Hologic, Marlborough, Massachusetts), ethanol fixed and dried overnight at room temperature. Heat-induced epitope retrieval (95°C) was performed in retrieval-buffer (Tris/EDTA-Puffer pH 9.0 - Roche, Basel, Switzerland) for 20 min, and nonspecific protein binding sites were blocked by incubation in 3 % BSA (bovine serum albumin)-PBS (phosphate buffer solution) (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) for 30 min at room temperature. Subsequently, the coating was carried out with the primary antibody cocktail (Ki67 (Dako Agilent Technologies, Santa Clara, California) 1 :600 and SEC62 (Institute for Medical Biochemistry and Molecular Biology - Saarland University Medical Center, Homburg, Germany) 1 :400 in 1 % BSA-PBS solution) for 60 min at 37 C. Slides were coated with visualization reagents Alkaline Phosphatase (Roche, Basel, Switzerland), horseradish peroxidase (Roche, Basel, Switzerland), then with DAB (3,3'-diaminobenzidine) substrate-chromogen solution (Roche, Basel, Switzerland), followed by fast-red chromogen solution (Roche, Basel, Switzerland) using the CINtecPLUS® kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. This lead to a red and brown staining at the SEC62 and Ki67 antigen sites, respectively. This step was followed by a counterstaining with hematoxylin (alcohol free - Sigma Aldrich Chemie GmbH, Taufkirchen, Germany). The preparations underwent a 2-step mounting procedure, first by using an aqueous mounting medium, followed by mounting step with Entellan (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany).

Interpretation of the SEC62/Ki67 dual stain

[106] Sec62/Ki67 dual stain was considered as positive if double immune-reactive squamous cells were present. As positive were considered the cells with a cytoplasmic red (SEC62) and a nuclear brown (Ki67) staining. Cases without dual positive squamous cells or if dual stain occurred only in glandular cells were considered as negative.

Statistical Analysis

[107] For statistical analysis, a chi square test and t-test Fishers exact test was used with SPSS software, version 20 (IBM, Chicago, Illinois). P < 0.05 was considered statistically significant (a=0.05).

Results

[108] Figure 1 shows exemplarily the immunocytochemical features of (A) negative Sec62/Ki67 dual stain, as well as (B) in dual stain positive specimens Sec62 immunostaining intensity grade 1 in, (C) 2, (D) 3 and (E) 4. A total of 100 cases with available CINtec® PLUS immunocytochemical results and histology (except 2 NILM and one AG-NOS case) were analyzed in this study. Of the 100 patients, all (100%) CIN3+ cases showed a SEC62/Ki67 positivity, while low grade lesions and normal cells were negative (Table 1 ). p16/KI67 dual stain was also positive in all CIN3+ cases and negative in normal cells. 2 of 12 (16.7%) LSIL samples were p16/KΪ67 positive (Table 2). Detailed information on the cytological diagnosis and staining results are delineated in Table 1 and Table 2.

Table 1 : Sample characteristics according to Sec62/Ki67 dual stain results. (NILM - negative for intraepithelial lesion or malignancy; AGC-NOS - atypical glandular cells not otherwise specified; ASC-US - atypical squamous cells of undetermined significance; LSIL - low grade squamous intraepithelial lesion; ASC-H atypical squamous cells - cannot exclude HSIL; HSIL - high grade squamous intraepithelial lesion; SCC - squamous cell carcinoma)

Table 2: Sample characteristics according to p16/Ki67 dual stain results. (NILM - negative for intraepithelial lesion or malignancy; AGC-NOS - atypical glandular cells not otherwise specified; ASC-US - atypical squamous cells of undetermined significance; LSIL - low grade squamous intraepithelial lesion; ASC-H atypical squamous cells - cannot exclude HSIL; HSIL - high grade squamous intraepithelial lesion; SCC - squamous cell carcinoma)

[109] Since Sec62/Ki67 dual stain can be positive in reactive proliferative endocervical glands, the assessment of cell morphology is, however, indispensable for the evaluation of Sec62/Ki67 dual staining to avoid false positive findings due to reactive endocervical cells.

[110] In our current study in dual positive samples we found high Sec62 reactivity with the CIN3 and even higher Sec62 reactivity in squamous cell carcinoma (SCC), but no correlation with the degree of dysplasia. However only SCC showed grade 4 Sec62 intensity in dual stain positive atypical cells (Fig 2.).

[111] Using CIN2+ and CIN3+ lesions, the sensitivities, specificities, and predictive values for Sec62/Ki67 dual stain and p16/KΪ67 dual stain were calculated and are listed in Table 3. The sensitivity for the Sec62/Ki67 for the detection of underlying CIN2+ was 94.37 % and was 100 % for detecting CIN3+. The corresponding specificities were 100 % for CIN2+ and 84.09 % for CIN3+. The p16/KΪ67 dual stain demonstrated similar sensitivities for detecting CIN2+ (95.52 %) and CIN3+ (100 %). When examining the specificities, p16/KΪ67 performed with lower values (CIN2+ 93.94 %; CIN3+ 77.27 %) probably due to positivity in LSIL cases. The positive (PPV) and negative predictive values (NPV) for detecting an underlying CIN2+/CIN3+ lesion using the Sec62/Ki67 cytology were 100 %/88.89 % (PPV) and 89.20 %/100 % (NPV), respectively, and were 96.97 %/84.85 % (PPV) and 91.18 %/100 % (NPV), for the p16/Ki67 dual stain.

Table 3: Sensitivity, specificity, positive and negative predictive values of Sec62/Ki67 and p16/Ki67 dual stain.

[112] Sec62/Ki67 showed a higher specificity for CIN3+ lesions (Table 3.). Since in cases with spontaneous regression of even in untreated CIN3 are known, ideally only those patients should be operated where without surgery a cervical carcinoma would actually develop (McCredie 2008). This would significantly reduce the number of operations performed and their complications (miscarriage, premature birth). A recent study by Habbema et al. estimated the harms of cervical cancer screening strategies in the US. In 2007 36 million Pap smears showed 2.3 million abnormal results, leading to 1.5 million biopsies 300000 treatments due to precancerous lesions, estimated to leading to 5000 preterm births (Habbema 2017). The development of new biomarkers as Sec62/Ki67 dual stain can achieve in addition to higher oncological safety and reduction of serious obstetrical complications (Habbema 2017). Goal of the establishment of new additional methods to the established screening with cytology and/or HPV test is to minimize the possible damage by false positive results (Habbema 2017).

[113] Sec62/Ki67 dual stain immunocytochemistry is a promising tool for cytologists in the interpretation of high-grade squamous lesions on cytological specimens and to better assess their risk of progression into SCC. Further studies are needed to evaluate the role of this new test in the routine cervical cancer screening, for example in the triage of ASCUS and ASC-H cytology.

Example 2: Dual staining of Sec62 and Ki-67 in vulvar samples

Materials and methods

Patients and LBC samples

[114] Residual cytological material from ThinPrep (Hologic, Marlborough, Massachusetts, USA) LBC samples from 45 patients who underwent colposcopy was available. All patients gave written consent for the use of their cytologic samples according to the Declaration of Helsinki, and the study protocol was approved by the local ethical review board.

Vulvar cytology

[115] The samples were collected with a Vulvar Sampler (Rovers Medical Devices, Oss, Netherlands) according to the manufacturer’s instructions (van den Einden 2012). The lesions were brushed three times at the punctum maximum of the lesion. Slides were prepared with a T2000 processor (Hologic), Papanicolaou stained according to the standard protocol and subsequently assessed by two experienced cytotechnologists. Based on the Bethesda System, the following cytological diagnoses were made: normal/vulvitis for benign cellular changes, LSIL— low-grade squamous intraepithelial lesion, and HSIL— high-grade squamous intraepithelial lesion, suspicious for SCC (van den Einden 2012; Jimenez-Ayala 2002).

Colposcopy, histology, p16/Ki67 dual stain

[116] Colposcopic and histologic findings were reviewed. Colposcopy was performed according to the colposcopic nomenclature for vulvar lesions from the International Federation for Cervical Pathology and Colposcopy (Bornstein 2012). Except for 9 cases with normal or minor colposcopic findings, histology was performed (in 18 cases, target biopsy only; in 18 cases, target biopsy followed by complete excision). P16/KΪ67 dual staining was performed in 27 cases with residual liquid-based-cytology (LBC) material after Sec62/Ki67 dual staining using a CINtec PLUS kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

Sec62/Ki67 dual stain in vulvar cytology

[117] To establish the Sec62/Ki62 dual stain in vulvar cytology, the Sec62 and Ki67 immunostains were individually tested with different antibody dilutions (1 :400, 1 :600, 1 :800) as already described in cervical cytology (submitted). The Sec62 staining in the 1 :400 dilution and the Ki67 staining in the 1 :600 dilution were successful. This step was followed by a sequential dual-staining procedure in different orders (Sec62 followed by Ki67 and Ki67 followed by Sec62) and with different dilutions (1 :400, 1 :600, 1 :800) in all possible combinations. Finally, simultaneous staining was tested with antibody cocktails with different dilutions of each antibody. In addition, individual steps of staining (fixation, unmasking, blocking) were performed with different methods to achieve the best possible results.

[118] Slides were prepared with a T2000 processor (Hologic), ethanol fixed and dried overnight at room temperature. Heat-induced epitope retrieval (95 °C) was performed in retrieval buffer (Tris/EDTA-buffer solution pH 9.0— Roche, Basel, Switzerland) for 20 min, and nonspecific protein binding sites were blocked by incubation in 3% BSA (bovine serum albumin)-PBS (phosphate buffer solution) (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) for 30 min at room temperature. Subsequently, coating was carried out with the primary antibody cocktail (Ki67 (Dako Agilent Technologies, Santa Clara, California, USA) at 1 :600 and Sec62 (Institute for Medical Biochemistry and Molecular Biology— Saarland University Medical Center, Homburg, Germany) at 1 :400 in 1% BSA-PBS solution) for 60 min at 37 °C. Slides were coated with the visualization reagents alkaline phosphatase (Roche, Basel, Switzerland), horseradish peroxidase (Roche), DAB (3,3'-diaminobenzidine) substrate- chromogen solution (Roche), and fast-red chromogen solution (Roche) using a CINtec PLUS kit (Roche) according to the manufacturer’s instructions. This process led to red and brown staining at the Sec62 and Ki67 antigen sites, respectively. This step was followed by counterstaining with hematoxylin (alcohol free— Sigma Aldrich Chemie GmbH). The preparations underwent a 2-step mounting procedure, first with an aqueous mounting medium, followed by a mounting step with Entellan (Sigma Aldrich Chemie GmbH) (Takacs 2018).

Interpretation of the Sec62/Ki67 dual stain

[119] Sec62/Ki67 dual staining was considered positive if double immunoreactive squamous cells were present. We considered cells with cytoplasmic red (Sec62) and nuclear brown (Ki67) staining to be positive (Fig. 3). Cases without dual positive squamous cells were considered negative.

P16/KΪ67 dual stain in vulvar cytology

[120] A CINtec PLUS kit (Roche, Basel, Switzerland) was used according to the manufacturer’s instructions. Positivity was defined by cytoplasmic brown (p16) and nuclear red (Ki67) staining (Fig. 4).

Statistical analysis

[121] For the statistical analysis, Chi-square test and Fisher’s exact test were used with SPSS software, version 25 (IBM, Chicago, Illinois). P values of < 0.05 were considered as statistically significant (a = 0.05).

Results

[122] This analysis included 45 cases. Cytological and colposcopic findings were categorized as“benign”— Normal/LSIL,“suspicious for VIN”— HSIL and“suspicious for malignancy”— SCC for statistical evaluation (Table 4). In 9 cases with normal colposcopy, no histology was available; in the remaining 36 cases, histology (only punch in 18 cases and punch and complete excision in 18 cases) was performed.

[123] Of the 45 patients, all (100%) vulvar intraepithelial neoplasia (VIN)/SCC cases showed Sec62/Ki67 positivity, while all (100%) low-grade lesions and normal cells were negative (Table 5). R16/KΪ67 was also positive in all VIN/SCC cases and negative in normal cells (Table 5). In Sec62/Ki67 positive cases, a different Sec62 staining intensity could be observed (Fig. 3). Every case with a Sec62 staining intensity of 3-4 was revealed as a malignancy; four cases with malignancy showed a lower (1-2) staining intensity, and none of the SCC cases was negative with the Sec62/Ki67 dual stain (Table 5). Detailed information on the histological, cytological findings and staining results are delineated in Table 5.

[124] To enable a comparison, despite the small sample size, we calculated the sensitivity and specificity of different diagnostic methods for detecting VI N and SCC (Table 6). Since all cases with normal findings or low-grade lesions of the vulva were Sec62/Ki67 and p16/KΪ67 negative and all VIN + findings were positive, we were able to calculate a 100% sensitivity and specificity for these methods. One case was overrated by cytology, and punch biopsy missed a case of VIN; consequently, cytology had a specificity of 95.7%, and punch biopsy had a sensitivity of 95.5%. In the detection of SCC, the sensitivity of the Sec62/Ki67 and p16/KΪ67 double-staining methods was 100%, but since all VIN cases were positive, the specificity was lower (85.2% and 76.9%). All cases with highly intensive Sec62 staining were carcinomas, leading to the highest specificity for the identification of malignancy among the methods studied. Table 6: Sensitivity and specificity of vulvar cytology, Sec62/Ki67 dual staining, and p16/KΪ67 dual staining for the detection of VIN and vulvar cancer

REFERENCES

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Claims

1. A method for detecting high grade dysplasia in a sample obtained from a subject, said sample comprising cells, said method comprising

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample indicates that said sample comprises cells characterized by high grade dysplasia.

2. The method of claim 1 , wherein the high grade dysplasia is a CIN3 (cervical intraepithelial neoplasia grade 3) lesion, a CIN 3+ lesion, a VIN3 (vulvar intraepithelial neoplasia grade 3), a VIN3+ lesion or carcinoma in situ (CIS).

3. The method of claim 1 or 2, wherein the absence of Sec62 and Ki67 in the cells is indicative for a CIN1 lesion, CIN2 lesion, CIN2+ lesion or normal cervical epithelium/NILM (negative for intraepithelial lesion or malignancy).

4. A method for assaying the potential for the development of a carcinoma in a sample obtained from a subject, said sample comprising cells, said method comprising

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for a high potential for the development of a carcinoma.

5. The method of any one of claims 1-4, wherein the sample is obtained from the urogenital tract, the anus or the otolaryngological regions.

6. The method of any one of claims 1-5, wherein the sample is obtained from the cervix or the vulva.

7. The method of any one of claims 1-6, wherein the sample is a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine.

8. The method of claim 7, wherein the swab sample is a Pap smear sample.

9. The method of any one of claims 1-8, wherein the subject is human.

10. The method of claim 9, wherein the human is a pregnant woman.

1 1. The method of claim 9 or 10, wherein the subject is positive in a Pap smear test and/or positive for biomarkers p16 and Ki67.

12. The method of any one of claims 1-1 1 , wherein the detection is performed by histology, DNA analysis, RNA analysis or protein analysis.

13. The method of any one of claims 1-1 1 , wherein the detection is carried out by immuno- staining.

14. The method of any one of claims 1-13, wherein the presence of Sec62 and Ki67 in cells of said sample is indicative for the necessity of a biopsy and/or colposcopy, whereas the absence of Sec62 and Ki67 is indicative that routine testing should be performed.

15. The method of any one of claims 1-14, wherein the detection of Sec62 and Ki67 is performed in squamous cells only.

16. The method of any one of claims 1-15, wherein the detection of Sec62 and Ki67 is not performed in reactive glandular cells.

17. A method for verifying whether a sample, which was determined to contain cells with high grade dysplasia, obtained from a subject indeed contains cells with high grade dysplasia, said sample comprising cells, said method comprising:

(i) detecting Sec62 and Ki67 in cells of said sample,

wherein the presence of Sec62 and Ki67 in cells of said sample indicates that the sample indeed contains cells with high grade dysplasia.

18. Use of agents for the specific detection of Sec62 and Ki67 in an in vitro method for the detection of high grade dysplasia in a sample obtained from a subject.

19. Use of agents for the specific detection of Sec62 and Ki67 for the prevention of a false- positive result of a sample obtained from a subject, wherein the false-positive result is a false classification of said sample, which was determined to contain high grade dysplasia cells.

20. A kit for detection high grade dysplasia in a sample comprising means for the specific detection of Sec62 and Ki67.