EP3314019A1

EP3314019A1 - Improved diagnostic method for hpv-induced cancer and precancerous lesion screening and diagnosis

Info

Publication number: EP3314019A1
Application number: EP16739420.4A
Authority: EP
Inventors: Sigrun SMOLA
Original assignee: Universitaet des Saarlandes
Current assignee: Universitaet des Saarlandes
Priority date: 2015-06-25
Filing date: 2016-06-27
Publication date: 2018-05-02
Also published as: WO2016207414A1

Abstract

The present invention relates to methods for diagnosis and/or prognosis of a HPV- induced precancerous lesion or a HPV-induced cancer. Also provided are methods for detecting cells of the transition zones zone of the cervix uteri, anorectal region, oropharynx, penis or vulva. In addition, the present invention encompasses uses of LGR6, LGR5 and/or LGR4 and kits comprising means for detecting these marker(s).

Description

Improved diagnostic method for HPV-induced cancer and precancerous lesion screening and diagnosis

FIELD OF THE INVENTION

The present invention relates to methods for diagnosis and/or prognosis of a HPV- induced precancerous lesion or a HPV-induced cancer. Also provided are methods for detecting cells of the transition zones of the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx or vulva. In addition, the present invention encompasses uses of LGR6, LGR5 and/or LGR4 and kits comprising means for detecting these marker(s).

DESCRIPTION

[001] Cervical cancer is the fourth most common cancer in women worldwide with 528,000 new cases and 266,000 deaths in 2012 (http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx?cancer=cervix). It is a rare consequence of persistent human papillomavirus (HPV) infection. Thus, human papillomavirus is regarded as a necessary cause of invasive cervical cancer worldwide. In addition, HPV has an etiological role for cancers of the penis, vulva, vagina, anus, conjunctiva and oropharynx (particularly base of the tongue and tonsillar cancer) (Humans IWGotEoCRt. A Review of Human Carcinogens. Part B: Biological Agents, Chapter 6, Human papillomaviruses IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France: IARC; 2009. p. 255- 313).

[002] HPV infection is a common event with an estimated risk of incident HPV infection (e.g. genital HPV infection) of more than 60% over 5 years (Baseman and Koutsky (2005) "The epidemiology of human papillomavirus infections." J Clin Virol; 32 Suppl 1 :S16-24). However, about two thirds of the HPV infections (e.g. genital HPV infection) are cleared within 1 year (Rodriguez et al. (2008) "Rapid clearance of human papillomavirus and implications for clinical focus on persistent infections." J Natl Cancer Inst; 100:513-7) and estimates on long-term persistent infections range from 4-10% (Bosch et al. (2013) "Comprehensive control of human papillomavirus infections and related diseases." Vaccine; 31 Suppl 7:1-11 -31 ). It is assumed that most HPV infections are "productive infections" leading to new infectious particles, while only a minority of infections become "transforming" with the potential to progress to cancer. To date, the conditions under which HPV infections become "transforming" have been unclear.

[003] With regard to genital HPV infection such productive infections were supposed to result from infections of basal cells of the ectocervical epithelium. For a long time infection of reserve cells located beneath the endocervical columnar epithelium close to the cervical squamocolumnar junction were suspected to play a central role in cervical intraepithelial neoplasia (Smedts et al. Basal-cell keratins in cervical reserve cells and a comparison to their expression in cervical intraepithelial neoplasia. Am J Pathol 1992;140:601 -12; Martens et al. Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal. Int J Gynecol Pathol 2009;28:381 -8). Recently, it has been suggested that infection of a discrete population of cuboidal squamocolumnar junction cells characterized by a set of markers including CD63 and keratin 7 may be implicated in the pathogenesis of cervical cancer (Herfs et al. (2012) "A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer." Proc Natl Acad Sci U S A; 109:10516-21 ).

[004] Based on the findings of the pathogenesis of cervical cancer a broadly used screening procedure is based on the conventional or liquid-based Papanicolau cytological test "Pap test, Pap smear". It is in use as sole test, as a co-test, i.e. together with HPV-testing, or as a reflex test after HPV-testing. Depending on the cytological abnormalities detected, further clinical procedures are being defined.

[005] However, it has also been found that cancers, such as cervical cancers actually arise in the so-called transition zone- where squamous and columnar epithelium meet. These transition zones (also called transformation zones) are located at different body sites, which are also susceptible to HPV-induced cancer, including the endo-ectocervix junction, anorectal junction/anal canal, cornea- conjunctiva junction and the esophagogastric junction (McNairn and Guasch (201 1 ) "Epithelial transition zones: merging microenvironments, niches, and cellular transformation." Eur J Dermatol; 21 Suppl 2:21 -8). Another type of transition zones can also be detected e.g. in vulva and penis.

[006] Therefore, the goal of the present invention is the detection/prognosis/diagnosis of transition zones but also precancerous lesions or cancers with high sensitivity and specificity. This has important consequences for the management of the patient, since precancerous lesions and cancer should be treated whereas overtreatment of a patient should be avoided.

[007] The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures.

[008] The above being said, the present invention relates to a method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample, wherein the detection of both, LGR6, LGR5 and/or LGR4 and HPV in said sample is indicative of a HPV-induced precancerous lesion or a HPV-induced cancer.

[009] The present invention also provides for a method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample, wherein the detection of LGR6, LGR5 and/or LGR4 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV-induced cancer.

[010] In addition, the present invention relates to a kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4. In some embodiments, the kit further comprises a mean for the detection of HPV.

[011] The present invention also relates to a use of LGR6, LGR5 and/or LGR4 for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the use further comprises detecting human papilloma virus (HPV). [012] Furthermore, the present invention relates to an in vitro method for detecting cells of a transition zone in a sample, the method comprising

(i) detecting LGR6, LGR5 and/or LGR4 in said sample, wherein the presence of LGR6, LGR5 and/or LGR4 indicates that the sample comprises cells of the transition zone, whereas the absence of LGR6, LGR5 and/or LGR4 indicates that the sample does not comprise cells of the transition zone; and wherein the transition zone is located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.

[013] The present invention additionally relates to a use of LGR6, LGR5 and/or LGR4 for the reduction of false-negative results of samples obtained from cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.

[014] Moreover the present invention relates to a use of LGR6, LGR5 and/or LGR4 for the detection of cells of a transition zone in a sample, wherein the transition zone is located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.

[015] The figures show:

[016] Figure 1. Shown is the transition zone (or transformation zone; TZ) of a normal cervix uteri in comparison to the ectocervical tissue. The transition zone is an area, where squamous and columnar epithelium meet. It is an area of actively maturing epithelium between the current squamocolumnar junction (SCJ) and the original squamous epithelium. In particular, in this area columnar cells are constantly changing into squamous cells. In premenopausal women, the transition zone is fully located on the ectocervix. After menopause the transition zone may move partially, and later fully, into the cervical canal.

[017] (A) and (B) were stained with anti-LGR4 Ab (1 :300), (C-E) with anti-LGR5 Ab (1 :300-1 :400), (F-l) with anti-LGR6 Ab (1 :400), (J) with anti-CD63 Ab (1 :2000), (K-N) were double-stained with anti-LGR6 Ab (1 :500) and anti-p63 (1 :1500). In Figure 1 (A), (C), (D), (F), (H), (I), (J) and (K) the TZ also comprises a metaplasia. While in Figure 1 (B), (E) and (G) a normal ectocervix is shown.

[018] Stainings show that LGR4 (A), LGR5 (C, D) and LGR6 (F, H, I) are expressed in subcolumnar and subglandular reserve cells, immature metaplasia, in some but not all columnar cells within the TZ and that LGR4 (B), 5 (E) and 6 (G) are not expressed in normal ectocervix. Immature squamous metaplastic cells of the immature metaplasia as described herein are of parabasal/basal type. They are characterized by a relatively high N/C ratio (N/C = nuclear-cytoplasmic ratio) and a reduced cytoplasm. This reserve cell hyperplasia is the first step of the squamous metaplastic process.

[019] Recently, it has been suggested that infection of a discrete population of cuboidal squamocolumnar junction cells characterized by a set of markers including CD63 and keratin 7 may be implicated in the pathogenesis of cervical cancer (Herfs et al. (2012) "A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer." Proc Natl Acad Sci U S A; 109:10516-21 ). However, staining with CD63 shows that it is expressed in all endocervical glands and columnar cells of the transition zone and endocervix (J). CD63 is not expressed in reserve cells or metaplastic epithelium but in the columnar cells that cover metaplastic epithelium (J).

[020] To further confirm that LGR4, LGR5 and LGR6 expression is present in reserve cells a staining with p63 was conducted. p63 is expressed in reserve cells but also in basal cells of the metaplasia, the squamous epithelium and squamous cell carcinomas.

[021] Basal cells of subcolumnar immature metaplasia stain double-positive for p63 and LGR6 (K). Subglandular reserve cells stain double-positive for LGR6 and p63 (L- N).

[022] Therefore, LGR4, LGR5 and LGR6 are furthermore expressed in subcolumnar and subglandular reserve cells, immature metaplasia, in some but not all columnar cells within the TZ and LGR4, 5 and 6 are not expressed in normal ectocervix.

[023] Figure 2. Shown is the extension of the TZ into the endocervical canal of a normal cervix uteri, which does not comprise a metaplasia. The TZ was stained with (A) anti-LGR4 Ab (1 :200), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-CD63 Ab (1 :2000).

[024] Stainings show that LGR4, 5, and 6 expression is strongest in columnar cells close to the squamocolumnar junction of the TZ and expression strongly decreases towards the endocervical canal (A-C). CD63, in contrast, is expressed in all endocervical columnar cells at equal intensity (D). [025] Figures 3-5. Shown are precancerous lesions such as high-grade squamous intraepithelial lesion (HSIL) of the cervix uteri. Importantly, the sections shown in each of Fig. 3-5 (A)-(F) represent subsequent sections. Therefore, these sections also allow for the evaluation, if the lesion also stains for different marker such as Ki67 and LGR4, 5 or 6 or Ki67 and CD63. (A) was stained with anti-LGR4 Ab (1 :350), (B) with anti-LGR5 Ab (1 :300-1 :500), (C) with anti-LGR6 Ab (1 :300-400), (D) with anti- pi 6 (1 :32-64), (E) with anti-Ki-67 (1 :1200) and (F) with anti-CD63 Ab (1 :2000). Staining for p16 was performed to detect transforming HPV infections. Often additionally a Ki-67 staining is performed. A cell positive for both Ki-67 and p16 will be infected with HPV. Furthermore, the presence of both markers in some cells detects the presence of a precancerous lesion such as HSIL.

[026] Stainings show that p16- and Ki-67-positive epithelial cells of the precancerous lesion (HSIL) express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in gland cells and in only some cell layers located above the LGR4-, 5-, 6-, p16- and Ki-67-positive HSIL. The majority of CD63-positive cells are however p16 low to negative and Ki-67 negative. This shows that precancerous lesions originate from LGR4, LGR 5 or LGR 6 positive cells but not from CD63- positive cells.

[027] Figure 6. Shown is a squamous cell carcinoma next to a precancerous lesion such as HSIL of the cervix uteri. (A) was stained with anti-LGR4 Ab (1 :350), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), (E) with anti-Ki-67 (1 :1200) and (F) with anti-CD63 Ab (1 :2000).

[028] Stainings show that p16- and Ki-67- positive epithelial cells of the squamous cell carcinoma and precancerous lesion (HSIL) express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in cells located above the LGR4-, 5-, 6-, p16- and Ki-67-positive precancerous lesion (HSIL). The majority of CD63- positive cells are p16 low to negative and Ki-67-negative.

[029] Figure 7. Shown is a squamous cell carcinoma of the cervix uteri. (A) was stained with anti-LGR4 Ab (1 :200), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), (E) with anti-Ki-67 (1 :1200) and (F) with anti- CD63 Ab (1 :2000).

[030] These further stainings confirm that p16- and Ki-67-positive epithelial cells of the squamous cell carcinoma express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in cells located beneath the LGR4-, 5-, 6-, p16- and Ki- 67-positive squamous cell carcinoma. The majority of CD63-positive cells are p16- negative and Ki-67-negative. Thus, LGR4, 5, and 6 can also be used to detect squamous cell carcinoma, while CD63 will not detect this type of cancer.

[031] Figures 8 and 9. Shown are adenocarcinomas of the cervix uteri. (A) was stained with anti-LGR4 Ab (1 :200), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), (E) with anti-Ki-67 (1 :1200) and (F) with anti- CD63 Ab (1 :2000).

[032] Stainings show that p16- and Ki-67-positive epithelial cells of the adenocarcinomas express LGR4, 5, and 6 and low levels of CD63. Thus, LGR4, 5, and 6 can also be used to detect adenocarcinomas.

[033] Figure 10. Shown is a CIN3 lesion in subsequent slices of the cervix uteri. Staining with anti-LGR4 Ab (1 :200), with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), with anti-CD63 Ab (1 :2000) was performed.

[034] Stainings show that p16-positive epithelial cells of the CIN3 lesion can also express LGR4, and 6 and p16. However, CD63 is not detected in this lesion. Thus, LGR4, 5, and 6 can also be used to detect CIN3 lesions.

[035] Figure 11. Sections of anorectal normal tissue and perianal carcinoma in situ were stained as described for cervical tissue. Shown is the normal anorectal transition zone (or transformation zone; TZ) (A) of a patient in comparison to anorectal glandular tissue outside the transformation zone (B) or normal anal mucosa outside the transformation zone (C). (A), (B) and were stained with anti-LGR6 Ab (1 :400). Stainings show that LGR 6 expression is strongest in epithelial cells close to the squamocolumnar junction of the TZ and in metaplastic cells. Anorectal glands or stratified epithelium outside the transformation zone are negative.

[036] Figure 12. Shown are precancerous lesions such as anal high-grade squamous intraepithelial lesion (AIN3, anal carcinoma in situ) from two different patients (A) and (B) stained with anti-LGR6 Ab (1 :400). In (B) it is shown that epithelial cells adjacent to the AIN3 also stain positive for LGR6.

[037] Figure 13. Normal human foreskin was stained as described for cervical tissue with anti-LGR6 Ab (1 :400). Stainings show that LGR6 expression is detected in mucosal stratifying epithelium. [038] Figure 14. Shown are immunocytochemical stainings of cervical ThinPreps samples from different patients with normal Pap smears. Samples were stained with anti-LGR4 Ab (1 :300), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :300). Photographs were taken at 200x magnification. Brown color indicates positive staining. Stainings show a LGR4- (A), LGR5- (B) or LGR6- (C) -positive epithelial cell indicating that the swabs were taken from the TZ.

[039] This invention aims at improving the early detection of HPV-induced cancers and precancerous lesions. The invention provides a more precise prognosis/diagnosis/detection of HPV-induced precancerous lesions with a high risk for progression to cancer and to separate these infections from those with a low risk of progression. This is possible because the stages in HPV-induced carcinogenesis e.g. cervical carcinogenesis include HPV infection; persistence, rather than clearance of the virus, linked to the development of a precancerous lesion and invasion. These are necessary stages since HPV-induced cancers (e.g. cervical cancers) are virtually impossible in the absence of intermediate progression to precancerous lesion. It may take only a few years until precancerous lesions arise, while progression to cancer requires more than 20 years on average (Vink et al. (2013) "Clinical progression of high-grade cervical intraepithelial neoplasia: estimating the time to preclinical cervical cancer from doubly censored national registry data." Am J Epidemiol;178:1 161 -9). This long time interval of progression to cancer allowed the establishment of cancer screening programs, such as cervical cancer screening programs, which have contributed to a strong reduction in cervical cancer, such as cervical cancer incidence and mortality in industrialized countries (Arbyn et al. (201 1 ) "Worldwide burden of cervical cancer in 2008." Ann Oncol; 22:2675-86).

[040] One broadly used screening procedure used in cervical cancer is based on the conventional or liquid-based Papanicolau cytological test "Pap test, Pap smear". It is in use as sole test, as a co-test, i.e. together with HPV-testing, or as a reflex test after HPV-testing. Depending on the cytological abnormalities detected, further clinical procedures are being defined. Notably, as described above, most precancerous lesions arise at the age of 25-30 years, about 10 years after sexual debut (Schiffman et al. (2007) "Human papillomavirus and cervical cancer." Lancet; 370:890-907). Currently, cytology is particularly recommended as the sole or primary screening method in women between 20 and 30 years of age, even in most countries where HPV-testing has been introduced for primary screening or co-testing in older age groups. The reason for this is the high prevalence of HPV including high risk HPV (hrHPV) types in this age group (Bruni et al. (2010) "Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings." J Infect Dis; 202:1789-99; Wright et al. (2015) "Primary cervical cancer screening with human papillomavirus: End of study results from the ATHENA study using HPV as the first-line screening test." Gynecol Oncol; 136:189-97).

[041] A single Pap test, however, has a low sensitivity to detect precancerous lesions, low reproducibility and accuracy (Nanda et al. (2000) "Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review." Ann Intern Med; 132:810-9; Fahey et al. (1995) "Meta-analysis of Pap test accuracy." Am J Epidemiol; 141 :680-9; Cuzick et al. (2006) "Overview of the European and North American studies on HPV testing in primary cervical cancer screening." Int J Cancer; 1 19:1095-101 ). The 3-years cumulative incidence rate of precancerous lesions (e.g. CIN3) or invasive cancer in cytology-negative women was 0.8% and the sensitivity for precancerous lesions (e.g. CIN3) or invasive cancer of cytology was less than 50% (Wright et al. (2015) "Primary cervical cancer screening with human papillomavirus: End of study results from the ATHENA study using HPV as the first-line screening test." Gynecol Oncol; 136:189-97). Pap test also has a low sensitivity as the sole reflex test for triage (sorting for further management) of HPV- positive women (Cox et al. (2013) "Comparison of cervical cancer screening strategies incorporating different combinations of cytology, HPV testing, and genotyping for HPV 16/18: results from the ATHENA HPV study." Am J Obstet Gynecol; 208:184 e1 - e1 1 ). Interlaboratory variation of liquid-based cytology varied from 42% to 73% Wright et al. (2014) "Interlaboratory variation in the performance of liquid-based cytology: insights from the ATHENA trial." Int J Cancer; 134:1835-43.

[042] Also, more than half of the women between 25-30 years with precancerous lesions (e.g. CIN2, CIN3) or invasive cancer had a negative cytology result (Wright et al. (2015) "Primary cervical cancer screening with human papillomavirus: End of study results from the ATHENA study using HPV as the first-line screening test." Gynecol Oncol; 136:189-97). Thus, a negative cytology result obtained with these tests does not imply that there is no precancerous lesion or no invasive cancer.

[043] In order to cover a broader range of women, self-sampling using a lavage or brush-based sampler has been suggested. However, such samples may contain less cervical cells with meaningful morphology and more vaginal cells resulting in a lower 10-19% sensitivity (Bosch et al. (2013) "Comprehensive control of human papillomavirus infections and related diseases." Vaccine; 31 Suppl 7:1-11 -31 ; Steenbergen et al. (2014) ..Clinical implications of (epi)genetic changes in HPV- induced cervical precancerous lesions." Nat Rev Cancer; 14:395-405). Thus, the problem of quality control is even aggravated, and respective methods have to be validated (Dijkstra et al. (2014) "Cervical cancer screening: on the way to a shift from cytology to full molecular screening." Ann Oncol; 25:927-35).

[044] More recently, HPV testing as a primary test with secondary triage testing or HPV co-testing has been introduced into screening programs in different countries. Most high-grade lesions (CIN2+) and about two-thirds of low-grade lesions (with microscopic signs of acute HPV-infection) are associated with carcinogenic HPV types (Schiffman et al. (201 1 ) "Human papillomavirus testing in the prevention of cervical cancer." J Natl Cancer Inst; 103:368-83). Several trials have demonstrated that hrHPV nucleic acid testing detects precancerous lesion (e.g. CIN2+ with about 20% and CIN3+ with a 30% higher sensitivities). A negative HPV test provides a higher reassurance of low cancer risk than cytology (Bosch et al. (2013) "Comprehensive control of human papillomavirus infections and related diseases." Vaccine; 31 Suppl 7:H1 -31 ).

[045] However, HPV testing cannot discriminate between transient and persistent infection, a prerequisite for cancer development. Thus, HPV testing has a 3-4% lower specificity to detect precancerous lesions (e.g. CIN2+), which is even worse if an ultrasensitive detection method is applied (Dijkstra et al. (2014) "Cervical cancer screening: on the way to a shift from cytology to full molecular screening." Ann Oncol; 25:927-35; Hesselink et al. (2008) "Comparison of GP5+/6+-PCR and SPF10-line blot assays for detection of high-risk human papillomavirus in samples from women with normal cytology results who develop grade 3 cervical intraepithelial neoplasia." J Clin Microbiol; 46:3215-21 ). This may result in a higher number of colposcopy referrals and overtreatment associated with potential side effects and higher costs.

[046] Due to the low sensitivity, a major consequence of using the Pap test for primary screening is frequent screening, which may, however, lead to an increase in false-positive results (Dijkstra et al. (2014) "Cervical cancer screening: on the way to a shift from cytology to full molecular screening." Ann Oncol; 25:927-35). Currently, there is no quality control available. Particularly, it is unclear whether negative cytology test results are truly negative, since there is no method that proves that the "right cells" prone to carcinogenesis are present in the sample and have been investigated.

[047] Notably, current HPV testing does not provide information, which part of the cervical mucosa, i.e. ectocervix, endocervix or the transition zone, is infected. Furthermore, it does not provide information whether this infection is transient or persistent. Knowledge of these factors would, however, increase test specificity and limit the number of colposcopy referrals and cases of overtreatment.

[048] The present inventors have surprisingly found that the presence of stem cell markers LGR4, 5 and/or 6 characterizes cells from which HPV-induced precancerous lesions and HPV-induced cancer such as cervical cancer originate. In particular, it was found that reserve cells as well as a subgroup of columnar cells in transition zones such as the transition zone of the normal cervix uteri express the stem cell markers LGR4, 5 and 6. These markers are also expressed in HPV-induced precancerous lesions such as cervical precancerous lesions (e.g. CIN3) and invasive cancer (squamous cell carcinomas as well as adenocarcinomas) such as cervical cancer (Figures and Examples as described herein). This confirms that these HPV- induced precancerous lesions as well as HPV-induced cancers originate from the LGR4, 5 and/or LGR6 expressing cells present in transition zones such as the transition zone of the cervix uteri.

[049] Data are additionally surprising because other publications indicated that infection of a discrete population of cuboidal squamocolumnar junction cells characterized by a set of markers including CD63 and keratin 7 is implicated in the pathogenesis of cervical cancer (Herfs et al. (2012) "A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer." Proc Natl Acad Sci U S A; 109:10516-21 ). However, as can be obtained from the figures and examples data demonstrate that reserve cells do not express CD63. Rather, CD63 expression is detected in cervical glands and cervical columnar cells including those located above immature metaplasia in normal cervical tissue. CD63 may also be detected in cell layers covering CIN2+ lesions. Importantly, CD63 expression is, however, not detected in the p16/Ki67-positive cell layers of CIN2+ and in the malignant cells of squamous cell carcinomas of the cervix uteri. Thus, LGR4, 5 and/or LGR6 expressing cells are a prerequisite for cervical carcinogenesis. On the contrary, CD63 is not a marker for the detection of precancerous lesions or squamous cell carcinogenesis of the cervix uteri.

[050] Notably, HPV-induced precancerous lesions and HPV-induced cancers can also originate from LGR4, 5 and/or LGR6 expressing cells, which are not located in a transition zone. Thus, LGR4, 5 and/or LGR6 expressing cells are also a prerequisite for carcinogenesis of cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar or cornea/conjunctiva. LGR4, 5 and/or LGR6 expressing cells can also be a prerequisite for carcinogenesis in the skin, head and neck (oropharynx, nasopharynx), oropharynx, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder as well as esophagus.

[051] Thus, the present invention provides a quality control reassuring that cells relevant for carcinogenesis are present in the sample investigated. Moreover, it can provide information that oncogenic HPV took place in cells relevant for carcinogenesis.

[052] Thus the invention helps (1 ) to assure that relevant lesions will be referred to colposcopy and/or treatment and (2) to prevent unnecessary surgical treatment and side effects for patients with lesions having a low risk of progression.

[053] The method of the present invention has important consequences for cancer and precancerous lesion screening, prognosis and diagnosis:

1 ) If LGR4, 5 and/or LGR6 expressing cells or respective markers cannot be detected in a sample, the investigation has to be judged as invalid. The lesion may be present but there are too few cells capable of carcinogenesis in the sample to provide a meaningful result.

2) If LGR4, 5 and/or LGR6 are detected in cells of a sample but do not show signs or markers of HPV infection, there is no need for surgical intervention/treatment and future control visits are sufficient.

3) If LGR4, 5 and/or LGR6 markers are detected in cells of a sample and show signs or markers of HPV infection and in particular high-risk HPV infection, there is a higher chance for the development of a high-grade precancerous lesion indicating (surgical) treatment. 4) With regard to samples obtained from the cervix uteri, anorectal region, penis, vulva, oropharynx or nasopharynx/oropharynx detection of LGR4, 5 and/or 6 in the sample (such as swabs, biopsies, self-samples) indicates that a sample has been taken in the transition zone (where stem cells prone to carcinogenesis reside). This is an important quality control reassuring that the relevant cells are present in the sample and being investigated.

5) Conversely, if the LGR4, 5 and/or 6 are lacking from the sample obtained from the cervix uteri, anorectal region, penis, vulva, oropharynx or nasopharynx/oropharynx the sample has not been collected in the region of interest, which is the transition zone. Therefore, it would need to be declared as invalid. Moreover, LGR4, LGR5, and/or LGR6-staining of conisation specimens will provide information whether the susceptible cells within the transition zone have been removed.

6) Notably, investigations using the LGR4, 5 and/or 6 are also suitable to exclude relevant HPV-induced precancerous lesions such as dysplasia, and cancers.

[054] Thus, the present invention relates to an in vitro method for detecting cells of a transition zone in a sample, the method comprising

It is also envisioned by the methods of the present invention, that the method is a method for the determination if a sample is suitable for HPV diagnosis. The present invention also encompasses that the methods of the present invention are suitable for HPV diagnostic procedures for the detection of HPV-induced precancerous lesions or HPV-induced cancers.

[055] A "transition zone" (TZ) as described herein is known to the skilled artesian and for example described in McCnairn and Guasch Epithelial transition zones: merging microenvironments, niches, and cellular transformation. Eur J Dermatol 201 1 ; 21 (Suppl. 2): 21 -28. A transition zone can span the junction between two types of epithelia. They are for example identifiable histologically, and can be found in numerous places including areas spanning the junctions between the esophagus and the stomach, between the stomach and the duodenum, the ileocecal junction between the final part of the small intestine and the beginning part of the large intestine, the junction of the endocervix and ectocervix, the cornea and the conjunctiva in the eye, and between the anal canal and the rectum.

[056] As mentioned herein transition zones can comprise two types of epithelia, namely the squamous epithelium and a columnar epithelium. The area of the transition zone is not only restricted to the area of the junction between the two types of epithelia. It is also envisioned by the present invention that the transition zone can comprise a squamo-columnar junction and is susceptible for HPV infection. It is further encompassed by the present invention that metaplasia as described herein can occur within the transition zone.

[057] The squamous epithelium can be a stratified squamous epithelium, which comprises reservoirs of stem cells that can replenish the tissue. This type of epithelium can for example be found in the gut or reproductive organs. Stratified squamous epithelium can be composed of several layers of cells, with the outermost layers having a characteristic flat appearance. The basal layer, which is in contact with the basement membrane can be mitotically active and can give rise to all the cells of the epidermis. The spinous layer can be the outer population of cells, the granular layer can be characterized by the presence of keratin granules, and the stratum corneum can consist of cornified keratinocytes.

[058] Stratified squamous epithelia may be either keratinized or non-keratinized. In humans, non-keratinized stratified squamous epithelium occurs in the cervix, cornea, most parts of the oral cavity, anal canal, and vagina, whereas, the skin represents keratinized stratified squamous epithelium. Simple epithelium consists of a single cell layer and may be either squamous, columnar or cuboidal. These types of epithelium are typically found in glands or other secretive tissues, including the stomach, intestines, rectum and glands.

[059] Additionally or alternatively the transition zone can also span two types of mucosa. Additionally or alternatively the transition zone can also span mucosa and/or skin. A mucous membrane or mucosa is a lining of mostly endodermal origin. It consists of an epithelium and an underlying lamina propria of connective tissue. This is for example the case for the transition zone of the vulva or penis as described herein.

[060] As further described herein, transition zones can be located at different body sites and can for example comprise the endo-ectocervix junction, anorectal junction, cornea-conjunctiva junction and also the esophagogastric junction, gastro-duodenal junction as well as ileocecal junction (McNairn and Guasch (201 1 ) "Epithelial transition zones: merging microenvironments, niches, and cellular transformation." Eur J Dermatol; 21 Suppl 2:21 -8). Thus, the term "transition zone" as used herein can include a transition zone comprising the endo-ectocervix junction and anorectal junction. The transition zone can also be located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. The transition zone can also be located in the cervix uteri and anorectal region.

[061] In this context it is noted that the HPV-induced cancer and/or HPV-induced precancerous lesion may also arise from any transition zone as described herein.

[062] Turning now to the specific transition zones. The transition zone can for example be located at in the cornea/conjunctiva. This transition zone spans parts of the stratified squamous epithelium of the cornea and the simple columnar epithelium of the conjunctiva. This transition zone is also referred to as limbus. This transition zone comprises the cornea-conjunctiva junction. It furthermore has a role in maintaining proper eye function and serves as a stem cell niche. This transition zone is also involved in the generation of HPV-induced precancerous lesions or cancers such as tumor formation in the cornea/conjunctiva.

[063] The transition zone can for example be located at in the cervix uteri. This cervical transition zone spans the squamo-columnar junction of the endocervix and ectocervix where the thicker stratified squamous epithelium of the ectocervix meets the glandular columnar epithelium of the endocervix. The transition zone of the cervix uteri is also known as the transformation zone. It is the common site of origin of precancerous lesions and cervical cancers triggered by human papilloma virus (HPV) infection.

[064] The transition zone can for example be located at in the anorectal region. The anorectal transition zone occurs in the area, where the stratified squamous epithelium of the anal canal meets the simple cuboidal epithelium of the rectum. The anorectal transition zone thus comprises epithelial cells. In particular, such epithelial cells can for example comprise reserve cells and/or columnar cells. Notably, the anal canal comprises the region where columnar and squamous epithelium meet. This transition zone comprises the anorectal junction/anal canal. This transition zone can occur above the dentate line and below the suprazonal irregular line, spanning a region of less than a centimeter. Also this region is susceptible to HPV infection. Furthermore, in this transition zone also precancerous lesions and cancers such as squamous cell carcinomas can be developed.

[065] The transition zone can for example be located in the nasopharynx/oropharynx. This transition zone spans the region, in which the stratified columnar epithelium of the nasopharynx, which changes into the stratified squamous epithelium of the oropharynx. The junction between these two types of epithelium are encountered by the junction of the roof and lateral walls.

[066] The transition zone can for example be located in the oropharynx. This transition zone includes transition zones in the tonsil and/or base of tongue. The transition zone of the oropharynx can be located in the tonsil. The transition zone of the oropharynx can be located in the base of tongue.

[067] The transition zone can for example be located in the vulva. This transition zone can be located subclitorally. In particular, this transition zone spans the clitoral and vaginal mucosa. Therefore, this transition zone comprises the junction between the vaginal and clitoral mucosa. This transition zone is for example described in Smeak and Turner (2014) "Treatment of clitoral hypertrophy containing an os clitoris and urethra in a young greyhound dog: A detailed surgical description" Can Vet J;55:836-840.

[068] The transition zone can for example be located in the penis. This transition zone can be e.g. located in the foreskin. However, the transition zone can also be located at another part of the penis such as e.g. glans penis. The transition zone of the penis can comprise epithelial cells. Therefore, this transition zone of the penis can comprise mucosal epithelium and/or skin. For example, the transitional zone of the penis can comprise mucosal stratifying epithelium. In particular, the transitional zone can, for example, comprise mucosal stratifying epithelium and skin of the foreskin. The transition zone of the penis can additionally or alternatively span inner and outer mucosal stratifying epithelium of the foreskin. [069] Cells of the transition zones as described herein can be detected by the detection of LGR6, LGR5 and/or LGR4. "LGR4" (GPR48) is also termed "Leucine- rich repeat-containing G-protein coupled receptor 4". The term LGR4 embraces any LGR4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[070] As such e.g. LGR4 can for example be LGR4 of Mus musculus (UniProt number: A2ARI4, version 1 last modified February 19, 2007) or a fragment or variant thereof; Rattus norvegicus (Rat) (UniProt number: Q9Z2H4; version 2, last modified June 25, 2013) or a fragment or variant thereof; Danio rerio (Zebrafish) (Brachydanio rerio) (UniProt number: E7FE13; version 1 , last modified March 7, 201 1 ) or a fragment or variant thereof; Pan troglodytes (Chimpanzee) (UniProt number: H2R5I5; version 1 , last modified March 20, 2012) or a fragment or variant thereof; Macaca mulatta (Rhesus macaque) (UniProt number: F6QK18; version 1 , last modified July 26, 201 1 ) or a fragment or variant thereof or Bos taurus (Bovine) (Uniprot number: F1 MLX5; version 3, last modified June 25, 2013) or a fragment or variant thereof. The LGR4 polypeptide can have a sequence of SEQ ID NO: 1 and can also comprise a fragment or variant thereof. The LGR4 nucleic acid molecule can also have a sequence of any of SEQ ID NO: 8 or 1 1 and can comprise a fragment or variant thereof.

[071] "LGR5" (GPR49, GPR67) is also termed "Leucine-rich repeat-containing G- protein coupled receptor 5". The term LGR5 embraces any LGR5 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[072] LGR5 can for example be LGR5 of Mus musculus (Mouse) (Uniprot number: Q9Z1 P4; version 2, last modified July 27, 2009) or a fragment or variant thereof; Rattus norvegicus (Rat) (Uniprot number: D4AC13; version 1 , last modified April 19, 2010) or a fragment or variant thereof; Pan troglodytes (Chimpanzee) (Uniprot number: H2Q6G9; version 1 , last modified March 20, 2012) or a fragment or variant thereof; Bos taurus (Bovine) (Uniprot number: F1 MT22; version 2, last modified November 15, 201 1 ) or a fragment or variant thereof. The LGR5 polypeptide can have a sequence of SEQ ID NO: 2, 3 or 4 and can also comprise a fragment or variant thereof. The LGR5 nucleic acid molecule can have a sequence of any of SEQ ID NO: 9, 12, 13 or 14 and can also comprise a fragment or variant thereof. [073] "LGR6" is also termed "Leucine-rich repeat-containing G-protein coupled receptor 6", respectively. The term LGR6 embraces any LGR6 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.

[074] LGR6 can for example be LGR6 of Rattus norvegicus (Rat) (Uniprot number: D3ZJU9; version 1 , last modified April 19, 2010) or fragments or variants thereof or Mus musculus (Mouse) (Uniprot number: Q3UVD5; version 1 , last modified October 10, 2005) or fragments or variants thereof or Danio rerio (Zebrafish) (Brachydanio rerio) (Uniprot number: P0DM44; version 1 , last modified June 25, 2013) or fragments or variants thereof. LGR6 polypeptide can also have a sequence of any of SEQ ID NO: 5, 6 or 7 and can also comprise fragments or variants thereof. The LGR6 nucleic acid molecule can also have a sequence of any of SEQ ID NO: 10, 15, 16 or 17 and can also comprise fragments or variants thereof.

[075] The term "nucleic acid molecule" when used herein encompasses any nucleic acid molecule having a nucleotide sequence of bases comprising purine- and pyrimidine bases which are comprised by said nucleic acid molecule, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences can include DNA, cDNA, genomic DNA, RNA, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[076] A variety of modifications can be made to DNA and RNA; thus, the term "nucleic acid molecules" can embrace chemically, enzymatically, or metabolically modified forms. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine.

[077] The term "polypeptide" when used herein means a peptide, a protein, or a polypeptide, which is used interchangeable and which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. Also encompassed by the invention are amino acids other than the 20 gene-encoded amino acids, such as selenocysteine.

[078] The term polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination; see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983), pgs. 1 -12; Seifter, Meth. Enzymol. 182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992); 48-62.

[079] A "variant" of the polypeptide of the present invention encompasses a polypeptide wherein one or more amino acid residues are substituted. For example, the substitution can be a conservative substitution compared to said polypeptide or to a polypeptide as depicted in any of SEQ ID NO: 1 -7. The variant can however still have the same functional properties as any of the polypeptides of SEQ ID NO: 1 -7. For example, in the case of LGR5 a variant may retain its functions as a WNT target gene. Such variants include insertions, inversions, repeats, and substitutions selected according to general rules known in the art which have no effect on the activity of the polypeptide compared to e.g. a polypeptide of SEQ ID NO: 1 -7.

[080] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, Science 247: (1990) 1306-1310. For example one strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

[081] A "variant" of a nucleic acid molecule of the present invention encompasses a nucleic acid molecule comprising a mutation. The mutation can be present with regard to any of SEQ ID NO: 8-17. Such mutations can include one or more point mutations, such as 1 , 2, 5, 10, 15, 20, 50 or more point mutations. A variant can also comprise insertions (addition of one or more nucleotides to the DNA RNA), such as 1 , 2, 3, 5, 6, or more insertions. Both, point mutations and insertions can be selected according to general rules known in the art, which can have no effect on the activity of the nucleic acid molecule compared to e.g. a nucleic acid molecule of SEQ ID NO: 8-17.

[082] Similarly, a "fragment" as used herein can be any nucleic acid molecule or polypeptide which comprises a deletion of 1 , 2, 3, 4, 5, 10, 20, 30 or more amino acid residues of any of SEQ ID NO: 1 -7 or a deletion of more than 1 , 2, 3, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or more nucleic acid bases compared to a nucleic acid molecule of any of SEQ ID NO: 8-17. The fragment can however still have the same functional properties as any of the polypeptides of SEQ ID NO: 1 -7 or the nucleic acid molecules of SEQ ID NO: 8-17. Such fragments can be selected according to general rules known in the art so as have no effect on the activity of the polypeptide as e.g. of a polypeptide of SEQ ID NO: 1 -7 or a nucleic acid molecule of SEQ ID NO: 8-17.

[083] Given that also variants and fragments of LGR6, LGR5 and/or LGR4 are encompassed by the present invention, the present invention also encompasses sequences which have a sequence identity of 80 %, 85 %, 90 %, 95 %, 97 %, 99 % or 100 % with any of the polypeptides/nucleic acid molecules of any of SEQ ID NO: 1 -17.

[084] In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid molecules or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 95 %, 96 %, 97 %, 98 % or 99 % identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 80 % to 95 % or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

[085] Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word size (W) of 28, an expectation (E) of 10, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, and an expectation (E) of 10. Furthermore, the BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used.

[086] For example, BLAST2.0, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments.

[087] The polypeptide can have the amino acid sequence encoded by a nucleic acid molecule as described herein e.g. in table 1 , such as any of SEQ ID NO: 8-17.

[088] Also, methods to detect LGR6, LGR5 and/or LGR4 are known to the skilled artesian. Methods to detect LGR6, LGR5 and/or LGR4 are also apparent for the skilled artesian when considering the sequences as depicted in Table 1 . In principle, the detection of LGR6, LGR5 and/or LGR4 can be performed by DNA, RNA or protein analysis.

[089] When analyzing DNA, such as DNA of any of SEQ ID NO: 8, 9, or 10 or a sequence having a sequence identity of 70 %, 80 %, 85 %, 90 %, 97 %, 99 % or 100 to any of SEQ ID NO: 8, 9, or 10. It can for example be determined if the DNA is transcribed/expressed by analyzing e.g. DNA methylation or histon modifications. Methods to detect DNA and also to detect if DNA is expressed are known to the skilled artesian and for example described in Wagner et al. (2014) "The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts." Genome Biology 2014, 15:R37 or Karlica et al. (2010) Histone modification levels are predictive for gene expression. PNAS vol. 107 no. 7, 2926-2931 .

[090] The detection of DNA (or methylation pattern or histone modifications of the DNA), such as DNA of any of SEQ ID NO: 8, 9, or 10 or a sequence having a sequence identity of 70 %, 80 %, 85 %, 90 %, 97 %, 99 % or 100 to any of SEQ ID NO: 8, 9, or 10 or HPV-DNA can be performed by any method. Such methods are known to the skilled artesian and for example described in Ghosh et al. (2006) "Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics." Mol. BioSyst; 2, 551-560. Exemplary methods for the detection of DNA (or methylation pattern or histone modifications of DNA) include PCR, southern blot, in situ hybridization or transcription-mediated amplification. Methylation pattern and histon modification patterns may also be analyzed with immunohistochemistry or immunocytology as described herein. Such methods can also include detection of LGR6, LGR5 and/or LGR4 cDNA. The DNA to be detected can also be a DNA of SEQ ID NO: 8, 9, or 10, which can also include the detection of fragments or variants thereof.

[091] Alternatively or additionally, LGR6, LGR5 and/or LGR4 but also HPV can also be detected by RNA analysis. Also these are standard techniques known to the skilled artesian. Exemplary methods for the detection of RNA are in situ hybridization, northern blot, RT-PCR or transcription-mediated amplification. The RT-PCR can also be a quantitative RT-PCR.

[092] In general, for the detection of DNA or RNA such as mRNA it may be useful to utilize one, two, three or more oligonucleotides (also called primers), which specifically hybridize to LGR6, LGR5 and/or LGR4 or fragments or variants thereof or HPV DNA or RNA such as mRNA. Such oligonucleotides can have a length of 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40 or more nucleic acid bases. Knowing the nucleic acid sequence of LGR4, LGR5 and LGR6 (e.g. SEQ ID NO: 8-17) or HPV DNA or RNA various oligonucleotide primers spanning the LGR4, LGR5 or LGR6 locus/RNA or HPV DNA or RNA may be designed e.g. in order to amplify the genetic material by Polymerase Chain Reaction (PCR).

[093] Conventional methods for designing, synthesizing, producing said oligonucleotide primers and performing PCR amplification may be found in standard textbooks, see, for example Agrawal (Ed.), "Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Methods in Molecular Biology, 20)", Humana Press, 1993; Innis et al. (Ed.), "PCR Applications: Protocols for Functional Genomics", Academic Press, 1999; Chen and Janes (Ed.), "PCR Cloning Protocols: From Molecular Cloning to Genetic", 2^nd edition, Humana Press, 2002. Once a nucleic acid molecule has been amplified, nucleotide structure can be analyzed by sequencing methods and compared to e.g. LGR4, LGR5 and LGR6 nucleic acid molecules such as of SEQ ID NO: 8-17 or fragments or variants thereof. Sequencing may be performed manually by any molecular biologist of ordinary skills or by an automated sequencing apparatus. These procedures are common in the art, see, for example, Adams et al. (Ed.), "Automated DNA Sequencing and Analysis", Academic Press, 1994; Alphey, "DNA Sequencing: From Experimental Methods to Bioinformatics", Springer Verlag Publishing, 1997.

[094] Thus, suitable oligonucleotide can hybridize to the nucleic acid sequence shown in any of SEQ ID NO: 8-17 or fragments or variants thereof or HPV RNA or DNA as described herein. Suitable oligonucleotides can be at least 65 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % complementary to the nucleic acid sequence shown in SEQ ID NO: 8-17 (or fragments or variants thereof) or HPV DNA or RNA. The term "hybridizes" as used in accordance with the present invention preferably relates to hybridizations under stringent conditions.

[095] It is also envisaged that the primer or pair of primers is labeled. The label may, for example, be a radioactive label, such as ³²P, ³³P or ³⁵S. In a preferred embodiment of the invention, the label is a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or a dye.

[096] Alternatively or additionally, LGR6, LGR5 and/or LGR4 and HPV can also be detected by the detection of the respective polypeptides. The polypeptide to be detected can also be a polypeptide of SEQ ID NO: 1 -7 or a polypeptide having a sequence identity of 80 %, 85 %, 90 %, 95 %, 97 %, 99 % to a sequence of any of SEQ ID NO: 1 -7. Such methods are known in the art. Exemplary methods to detect LGR6, LGR5 and/or LGR4 or HPV protein are immunohistochemistry, immunocytology, chromatographic methods or western blot.

[097] With immunohistochemistry 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. 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 LGR6, LGR5 and/or LGR4 protein. Thus, both immunohistochemistry and immunocytology can also comprise fluorescent or non-fluorescent immunohistochemistry and/or immunocytology. [098] Exemplary means to detect LGR6, LGR5 and/or LGR4 protein but also HPV protein 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.

[099] 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. Exemplary antibodies that can be used in the present invention include an anti-LGR4 polyclonal antibody (Sigma Aldrich), a monoclonal anti-LGR5 (Abeam; such as of clone: EPR3065Y), a monoclonal anti-LGR6 antibody (Abeam; such as of clone number EPR6874).

[100] 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).

[101] 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.

[102] 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 LGR4, LGR5 or LGR polypeptides but also proteins associated with HPV as described herein.

[103] 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.

[104] The methods of the present invention include that LGR6, LGR5 and/or LGR4 is detected in a sample. Additionally, also HPV such as a high risk HPV can be detected in the sample. HPV can further be detected in the same sample as LGR6, LGR5 and/or LGR4 or in a different (second) sample. The sample can be any sample, which is suitable for the methods of the present invention. For example, the sample can be a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine.

[105] An exemplary swab sample can be a pap (papanicolau) 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 uteri or anorectal region.

[106] The tissue sample can for example be a conisation sample. Cervical conization 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. In particular, it may be interesting if indeed LGR4, LGR5 and/or LGR6 positive cells of the transition zone have been removed. It is also possible to obtain a sample of a woman who has undergone conisation, to evaluate if cells of the transition zone remained/were not excised. For that e.g. a pap smear sample may be used.

[107] 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.

[108] The term "subject" can mean human or an animal. The subject can be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Preferably, a mammal is as 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 being.

[109] It also contemplated by the methods of the present invention that the method is a method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method further comprises

(ii) detecting human papilloma virus (HPV) in said sample,

wherein the detection of both LGR6, LGR5 and/or LGR4 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer. Likewise the present invention envisages a method for diagnosis and/or prognosis of a HPV- induced precancerous lesion or a HPV-induced cancer, wherein the method further comprises

(ii) detecting human papilloma virus (HPV) in said sample,

wherein the detection of LGR6, LGR5 and/or LGR4 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV-induced cancer. [110] In addition the present invention relates to a method for diagnosis and/or prognosis and/or detection of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample,

wherein the detection of both LGR6, LGR5 and/or LGR4 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer and

wherein the detection of LGR6, LGR5 and/or LGR4 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV-induced cancer. In the methods described herein the HPV can be a high risk HPV.

[111] The term "diagnosis" when used herein means the process of determining which disease or condition a subject has or is afflicted with. With regard to the present invention it can e.g. be detected if a subject has a HPV-induced precancerous lesion or a HPV-induced cancer. Information required for diagnosis is typically obtained from the subject to be diagnosed in the form of a sample. The sample can then be tested for the presence of LGR6, LGR5 and/or LGR4 and HPV and depending on the outcome the subject is diagnosed to have a HPV-induced precancerous lesion or a HPV-induced cancer or to not have HPV-induced precancerous lesion or a HPV-induced cancer. The subject can be diagnosed of having a precancerous lesion if the sample is positive for both, LGR6, LGR5 and/or LGR4 and HPV. The subject can be diagnosed of not having a HPV-induced precancerous lesion or not having a HPV-induced cancer if the sample is positive for LGR6, LGR5 and/or LGR4 and negative for HPV.

[112] In general, the sample in which LGR6, LGR5 and/or LGR4 and HPV are detected can be the very same sample (one sample). However, it can also be that LGR6, LGR5 and/or LGR4 and HPV are detected in different samples. Therefore, the present invention also encompasses more than one sample such as two, three or more samples to be analyzed. Furthermore, the presence of LGR6, LGR5 and/or LGR4 can be detected before further detecting HPV in a sample. It is also possible, that HPV is detected first in a sample and then the presence of LGR6, LGR5 and/or LGR4 is detected. It is further encompassed by the present invention that the presence of LGR6, LGR5 and/or LGR4 and HPV is detected at the same time in a sample. [113] It is further envisioned by the present invention that the subject or sample can be compared to a control. This can be done to determine if a derivation of the tested marker is present in the sample or not. The "control" can e.g. be a predetermined standard obtained from e.g. healthy individuals or a mean value obtained from many such as 2 or more different healthy subjects. The control can also be a sample of a healthy individual. It is also contemplated that the control can be a sample obtained from a subject not afflicted with a HPV-induced precancerous lesion or a HPV- induced cancer.

[114] Therefore, the methods of the present invention also encompass detection of increased levels of both, LGR6, LGR5 and/or LGR4 and eventually also HPV in a sample. The term "detecting" or detection of a marker such as LGR6, LGR5 and/or LGR4 as well as eventually HPV means measuring/analyzing the presence or absence of the measured marker as described herein. Also envisioned is the detection of decreased or no level of both, LGR6, LGR5 and/or LGR4 and HPV. Further envisioned is the detection of increased levels of LGR6, LGR5 and/or LGR4 and no HPV.

[115] The term "increased" or "upregulated" when used herein means that a certain marker (such as LGR6, LGR5 and/or LGR4, HPV or a cell cycle inhibitor) is expressed at a higher level compared to a control or a control level as described herein. The control can for example be a sample from a healthy subject, or a subject not afflicted with a HPV-induced precancerous lesion or HPV-induced cancer as described herein. In principle both protein expression or mRNA expression can be upregulated when compared to a control sample. For example, protein or mRNA expression level of marker of interest (such as LGR6, LGR5 and/or LGR4, HPV or a cell cycle inhibitor) a sample may be upregulated by 3 %, 5 % 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % or more when compared to the protein or mRNA expression level of a control sample.

[116] However, in the present invention such a comparison to a control can be dispensable, because the presence of both, LGR6, LGR5 and/or LGR4 and HPV in a sample already provides for a valuable diagnosis and/or prognosis and/or detection of a HPV-induced precancerous lesion or a HPV-induced cancer.

[117] In addition to the diagnosis of a HPV-induced precancerous lesion or a HPV- induced cancer, the methods of the present invention can also be used for the prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer. When used herein the term "prognosis" means the prediction of the likelihood of the development of a HPV-induced precancerous lesion or a HPV-induced cancer. Therefore, the prognosis can include assigning the likelihood of a future or a worsening of a HPV-induced precancerous lesion or a future HPV-induced cancer to e.g. a subject from which the sample has been obtained. Exemplary prognosis scores for early-stage squamous cell cervical cancer have already been established by Hellberg and Tot (Hellberg and Tot (2014) "Tumor marker score for prognostication of early-stage squamous cell cervical cancer." Anticancer Res; 34(2):887-92).

[118] Prognosis of a HPV-induced precancerous lesion and a HPV-induced cancer is possible because the stages in HPV-induced carcinogenesis e.g. cervical carcinogenesis include HPV infection and its persistence (no clearance of the virus), which is linked to the development of progressively worsening precancerous lesions, wherein the most severe precancerous lesion is a carcinoma in situ, that terminates in cancers. These stages usually occur since HPV-induced cancers (e.g. cervical cancers) are virtually impossible in the absence of intermediate progression via precancerous lesions.

[119] Thus, cancers, such as cervical cancers are a rare consequence of persistent human papillomavirus (HPV) infection (Walboomers et al. (1999) "Human papillomavirus is a necessary cause of invasive cervical cancer worldwide." J Pathol; 189:12-9). In this context it is noted that most HPV infections are "productive infections" leading to new infectious particles, while only a minority of infections become "transforming" with the potential to progress to cancer.

[120] In addition to the cervix uteri, HPV also has an etiological role for cancers of the penis, vulva, vagina, anus, conjunctiva, nasopharynx/oropharynx and oropharynx (particularly base of the tongue and tonsillar cancer) (Humans (2009) "A Review of Human Carcinogens. Part B: Biological Agents" Chapter 6, Human papillomaviruses IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France: IARC; 2009. p. 255-313). Thus, by the methods described herein a HPV- induced precancerous lesion or HPV-induced cancer as described herein can be prognosed. For example, HPV-induced precancerous lesions and/or HPV-induced cancers of the cervix uteri, penis, vulva, vagina, anus, conjunctiva (or cornea/conjunctiva), nasopharynx/oropharynx and oropharynx (particularly base of the tongue and tonsillar cancer) can be prognosed by the methods of the present invention. Also HPV-induced precancerous lesions and/or HPV-induced cancers of the cervix uteri, vulva, anus, nasopharynx/oropharynx and oropharynx (particularly base of the tongue and tonsillar cancer) can be prognosed by the methods of the present invention. HPV-induced precancerous lesions and/or HPV-induced cancers of skin, head and neck (oropharynx, nasopharynx), oropharynx, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder can also be prognosed/detected/diagnosed by the methods of the present invention. HPV- induced precancerous lesions and/or HPV-induced cancers of cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva can also be prognosed/detected/diagnosed by the methods of the present invention. In particular, HPV-induced precancerous lesions and/or HPV-induced cancers of cervix uteri and anus can be prognosed/detected/diagnosed by the methods of the present invention.

[121] Notably, cervical cancers (such as e.g. carcinoma) as well as cancers of the oropharynx/nasopharynx, oropharynx, anus/anorectal region, cornea/conjunctiva or vulva as described herein arise in the so-called "transition zones" comprising the junctions where squamous and columnar epithelium meet or where two different types of mucosa meet.

[122] The prognosis of HPV-induced cancer can for example be performed via the detection of a HPV-induced precancerous lesion. This is possible, because typically a precancerous lesion over time can progress into a carcinoma in situ, which still is considered a precancerous lesion. This lesion can further progress to become a cancer such as an invasive cancer. Additionally, or alternatively this can also be achieved via the detection of the presence or absence of a transforming infection. The detection of the presence or absence of a transforming infection can also allow for the prognosis of a precancerous lesion. Such a transforming infection includes the detection of HPV-positive or infected cell in a transition zone, preferably the transition zone of the cervix uteri. A transforming infection can also include the detection of HPV-positive or infected cell and LGR6, LGR5 and/or LGR4 in a sample. It can however also include the detection of a cell which is positive for LGR6, LGR5 and/or LGR4 and HPV. [123] The term "HPV-induced precancerous lesion" when used herein means any HPV-induced lesion that has the potential to develop into a cancer. A HPV-induced precancerous lesion is any precancerous lesion that is associated with or linked to an HPV infection. Such HPV-induced lesions are known to the skilled artesian and for example described in Schiffman and Wentzensen (2013) "Human papillomavirus (HPV) infection and the multi-stage carcinogenesis of cervical cancer." Cancer Epidemiol Biomarkers Prev; 22(4): 553-560. The HPV-induced precancerous lesion can be associated with high risk HPVs as described herein. For example, the HPV- induced precancerous lesion can be associated with HPV 16, HPV 18, HPV 31 and/or HPV 33.

[124] Methods to identify precancerous lesions are known to the skilled artesian. For example methods to detect oral cancer or precancerous lesions are described in Brad et al. (2002) "Day Oral Cancer and Precancerous Lesions. Cancer Journal for Clinicians." Volume 52, Issue 4, pages 195-215, Fuju Chang, et al. (1990) "Human papillomavirus (HPV) DNA in esophageal precancer lesions and squamous cell carcinomas from China. International Journal of Cancer." Volume 45, Issue 1 , pages 21-25, 15 January 1990) or precancerous lesions in head and neck cancer are e.g. described in Syrjanen (2005) "Human papillomavirus (HPV) in head and neck cancer." Journal of Clinical Virology. Volume 32, Supplement, pages 59-66.

[125] The HPV-induced precancerous lesion can also comprise dysplasia. "Dysplasia" when used herein refers to an abnormality of development or an anomaly of growth and differentiation of cells. In dysplasia cell maturation and differentiation can be delayed. The dysplasia can be an epithelial dysplasia, in which epithelial anomaly of growth and differentiation is detectable. Epithelial dysplasia can consist of an expansion of immature cells (such as cells of the ectoderm), with a corresponding decrease in the number and location of mature cells. Dysplasia can be indicative of an early neoplastic process. Dysplasia can also be indicative of an HPV-induced precancerous lesion.

[126] Methods of how a dysplasia can be detected are known to the skilled artesian and for example described in Collier et al. (2002) "Near Real-Time Confocal Microscopy of Amelanotic Tissue: Detection of Dysplasia in ex Vivo Cervical Tissue." Academic Radiology Volume 9, Issue 5, pages 504-512; Volgareva et al. (2004) "Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells." BMC Cancer; 4:58. For example, dysplasia may be detected via microscopically visible changes in cell morphology. As such anisocytosis (cells of unequal size), poikilocytosis (abnormally shaped cells), hyperchromatism (excessive pigmentation) and/or presence of mitotic figures (an unusual number of cells which are currently dividing) can be present in a dysplasia. Dysplasia of cells can be recognized in a pap smear or in another sample as described herein.

[127] The HPV-induced precancerous lesion can for example be a CIN lesion. "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 twothirds of the epithelium, with more marked nuclear abnormalities than in CIN1 . 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. The HPV- induced precancerous lesion may be a CIN2 or CIN3 lesion. The HPV-induced precancerous lesion can also be a CIN3 lesion. Accordingly, the HPV-induced precancerous lesion can also be a carcinoma in situ. The HPV-induced precancerous lesion 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 cytologic abnormality associated with distinctive HPV and histopathologic CIN 2+ detection rates." Am J Clin Pathol;141 (2):239-46.

[128] The HPV-induced precancerous lesion can also be a low grade or a high grade HPV-induced precancerous lesion. The risk of low grade HPV-induced 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 a low grade HPV-induced precancerous lesions can have a risk of developing cancer of not more than 50 %, 40 % 30 %, 20 %, 10 % 5 %, 3 %, 1 % 0 %.

[129] The low grade HPV-induced 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 p16 as described herein.

[130] A high grade HPV-induced precancerous lesion represents a more advanced progression towards malignant transformation. The risk of high grade HPV-induced precancerous lesion for transforming to cancer is high. This means that a subject with a high grade HPV-induced precancerous lesions has a risk of developing cancer of more than 20 %, 30 %, 40 % 50 %, 60 % 70 %, 80 %, 90 %, 95 %, 93 %, 97 % 99 % or 100 %. High grade HPV-induced precancerous lesions may be transforming infections or an HPV infection of cells of the transition zone such as a reserve cell, columnar cell or cuboid cell as described herein.

[131] A high grade HPV-induced precancerous lesion can for example be a high- grade squamous epithelial lesion. High grade squamous epithelial lesions are well known to the skilled artesian and have also been termed by the Bethesda System terminology and is 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 (CIN2), carcinoma in situ (CIN3) or CIN2, 3. The high grade squamous epithelial lesions may be a CIN3 or CIN2 lesion, wherein the CIN2 lesion can be positive for p16. The term CIN2+ when used herein means a precancerous lesion that is CIN2 or CIN3.

[132] Alternatively or additionally, the HPV-induced precancerous lesion can comprise high-grade squamous epithelial lesions such as CIN2+ lesions. The high- grade squamous epithelial lesion can also be a CIN3 lesion.

[133] The HPV-induced precancerous lesion can therefore be a carcinoma in situ (CIS). The CIS can for example be 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 HPV- induced precancerous lesion can also be an adenocarcinoma in situ or a squamous cell carcinoma in situ. It is also contemplated by the present invention that both an adenocarcinoma in situ and a squamous cell carcinoma in situ can be detected by the methods of the present invention.

[134] 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.

[135] For example, the HPV-induced precancerous lesion can also be a conjuntival intraepithelial neoplasia (CIN). These lesions 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.

[136] When the abnormal conjunctival epithelial cellular proliferation involves only partially the epithelium thickness it can be classified as mild CIN, a condition also called mild or moderate dysplasia. When it affects full thickness epithelium it can be called severe 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.

[137] The HPV-induced precancerous lesion 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.

[138] 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.

[139] 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 HPV-induced precancerous lesion can be a HSIL.

[140] The HPV-induced precancerous lesion can also be 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.

[141] 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 a HPV-induced precancerous lesion.

[142] The HPV-induced precancerous lesion 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,

[143] 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 are abnormal, while in VAIN3, the full thickness is affected. VAIN3 is also known as carcinoma in situ. Thus, the HPV-induced precancerous lesion can be a VAIN2 or VAIN 3 lesion. The HPV-induced precancerous lesion can be a VAIN3 lesion.

[144] 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.

[145] Mild dysplasia (grade I/SIN1 ) 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 11 l/SI N3) 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 lesion 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 characterised 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 keratinised surface layer. Thus, the HPV-induced precancerous lesion can be a SIN2 or SIN3 lesion. The HPV-induced precancerous lesion can also be a SIN3 lesion.

[146] 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.

[147] Considering the HPV-precancerous lesions as described herein, the precancerous lesion 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 HPV-induced precancerous lesion 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).

[148] In general, the HPV-induced precancerous lesion can be selected from the group consisting of HPV-induced precancerous lesions of the cervix uteri, anus, vagina, vulva, penis, oropharynx such as tongue or tonsillar cancer, oropharynx, cornea/conjunctiva or skin. The HPV-induced precancerous lesion can also be selected from skin, head and neck (oropharynx, nasopharynx), lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder. The HPV-induced precancerous lesion can also be selected from cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva.

[149] The methods of the present invention can also diagnose and/or prognose and/or detect a HPV-induced cancer. An HPV-induced cancer can be any cancer which is associated or linked with HPV infection. Such cancers are known to the skilled artesian and for example described in Haedicke and Iftner (2013) "Human papillomaviruses and cancer." Radiother Oncol; 108(3):397-402 and Rosales and Rosales (2014) "Immune therapy for human papillomaviruses-related cancers." World J Clin Oncol; 5(5):1002-19. HPV-induced cancers can for example be associated with high-risk HPV as described herein. HPV-induced cancers can also be associated with HPV 16 and HPV 18.

[150] For example, the HPV-induced cancer can be selected from the group consisting of HPV-induced cancers of the cervix uteri, anus, vagina, vulva, penis, oropharynx such as tongue or tonsillar cancer, oropharynx/nasopharynx, cornea/conjunctiva or skin. The HPV-induced cancer can also be selected from skin, head and neck (oropharynx, nasopharynx), lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder. The HPV-induced cancer can also be selected from cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva. Additionally, or alternatively the cancer can also be a HPV-induced cancer of which about 5 %, 10 %, 15 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % 99 % or 100 % of the respective cancer (cases) are linked to HPV. This means that e.g. 70-90 % of all oropharyngeal cancers (cancers of the back of the throat, including the base of the tongue and tonsils) can be linked to HPV or that about 80-90 % of all anal cancers can be linked to HPV.

[151] The person skilled in the art knows which type of cancer is associated with which type of HPV. This is also described in International Agency for Research on Cancer (2012) "Biological agents-A review of human carcinogens" IARC monographs on the evaluation of carcinogenic risks Vol. 100B (ISBN 978 92 832 13192) (this publication is obtainable from the webpage http://monoqraphs.iarc.fr/ENG/Monoqraphs/vol100B/mono1 OOB.pdf as of 29 May 2015). For example, oropharynx cancer such as tongue or tonsillar cancer can be associated with HPV 16 and HPV 18. Skin cancer may be associated with HPV 5, HPV 8, HPV 9, HPV 12, HPV 14, HPV 15, HPV 17, HPV 19, HPV 20, HPV 21 , HPV 22, HPV 23, HPV 24, HPV 25, HPV 36, HPV 37, HPV 38, HPV 46, HPV 47 or HPV 49. Skin cancer may also be associated with HPV 5 or HPV 8. Cancers of the cervix uteri, anus, vulva, vagina or penis can for example be associated with HPV 16. Cancers of the cervix uterus can also be associated with HPV 16, HPV 18, HPV 31 and/or HPV 45.

[152] The HPV-induced cancer can be a carcinoma and/or an invasive cancer. Exemplary carcinomas include adenocarcinoma, squamous cell carcinoma, a small cell carcinoma, a neuroendocrine tumor, a glassy cell carcinoma or a villoglandular adenocarcinoma. The HPV-induced cancer/carcinoma can be an adenocarcinoma or a squamous cell carcinoma. The methods of the present invention can provide for the detection of both an adenocarcinoma and a squamous cell carcinoma.

[153] For example, squamous cell carcinoma can also arise in stratified squamous epithelia. They can arise in epidermal keratinocytes and may occur in the skin, head and neck (oropharynx, nasopharynx), esophagus, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder. Thus, the HPV-induced cancer can occur in the skin, head and neck (oropharynx, nasopharynx), oropharynx, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder.

[154] The HPV-induced cancer can also be an invasive cancer. Such "invasive cancers" or "infiltrating cancers" are cancers that have spread beyond the layer of tissue in which it developed and is growing into surrounding, healthy tissues. For example, the cancer may transverse the basal membrane and become an invasive cancer. The basement membrane is present in every tissue of the human body, which is known to the skilled artesian (LeBleu et al. (2007) "Structure and function of basement membranes." Exp Biol Med (Maywood); 232(9):1 121 -9; Yurchenco (2010) Basement Membranes: Cell Scaffoldings and Signaling Platforms. Cold Spring Harb Perspect Biol a00491 1 ). The basement membrane can for example be a thin, extracellular membrane underlying epithelial tissue. Exemplary invasive cancers can be an invasive adenocarcinoma or an invasive carcinoma. In general, invasive cancers may express CD133. Therefore, invasive cancers can be detected by their expression of CD133 with e.g. immunocytology or immunohistochemistry. Upon further progression the invasive cancer may even form metastasis.

[155] The HPV-induced invasive cancer can be an invasive adenocarcinoma, invasive squamous cell carcinoma, invasive small cell carcinoma, invasive neuroendocrine tumor, invasive glassy cell carcinoma or invasive villoglandular adenocarcinoma. The HPV-induced cancer/carcinoma can be an invasive adenocarcinoma or an invasive squamous cell carcinoma. The methods of the present invention can provide for the detection of both an invasive adenocarcinoma and an invasive squamous cell carcinoma. The methods of the present invention can also provide for the detection of both an adenocarcinoma and a squamous cell carcinoma.

[156] The methods of the present invention provide for the diagnosis and/or prognosis as well as detection of a HPV-induced precancerous lesion or a HPV- induced cancer. For the prognosis and also for the diagnosis the absence or presence of a cell, which is capable of carcinogenesis can be detected. It is also encompassed by the present invention that one or more cells such as 2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, or more cells that are capable of carcinogenesis can be detected in a sample.

[157] This means that cells prone for transformation can be detected. Such cells may first give rise to a precancerous lesion and then progress into a cancer cell. The process of carcinogenesis is well known to the skilled artesian and for example described in Schiffmann and Wentzensen (2013) "Human papillomavirus (HPV) infection and the multistage carcinogenesis of cervical cancer." Cancer Epidemiol. Biomarkers Prev; 22(4): 553-560. In principle, any cell that is able to transformation/carcinogenesis is embraced by the present invention. The cell capable of carcinogenesis can for example be a cell located in a transition zone as described herein. [158] The cell capable of carcinogenesis may be a cell which undergoes metaplasia. The term "metaplasia" when used herein means the morphological and functional transformation of a tissue into another tissue with different morphology and function. It can for example include the reversible replacement of one differentiated cell type with another mature differentiated cell type. The change from one type of cell to another may generally be a part of normal maturation process, which can for example be seen in transition zones as described herein. However, metaplasia can also be caused by any suitable stimulus.

[159] A cell capable of transformation can also be a reserve cell, a columnar cell or a cuboidal cell. These cells are known to the skilled artesian and for example described in Herfs et al. (2012) "A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer." Proc Natl Acad Sci U S A; 109:10516-21 or Martenset al. (2007) "Reserve Cells in Human Uterine Cervical Epithelium Are Derived from Mullerian Epithelium at Midgestational Age" International Journal of Gynecological Pathology Volume 26, Issue 4, pp 463-468 or Martens et al. (2009) "Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal." Int J Gynecol Pathol;28:381 -8.

[160] The cell capable of carcinogenesis can also be a reserve cell or a columnar cell. The cell capable of carcinogenesis can also have the capacity to form spheres as described in Lopez et al. (2012) "Human Papillomavirus Infections and Cancer Stem Cells of Tumors from the Uterine Cervix." Open Virol J; 6: 232-240.

[161] Different methods are known to the skilled artesian to detect cells which are capable of carcinogenesis. Some of the methods are also described in Schiffmann M. and Wentzensen N. Human papillomavirus (HPV) infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiol. Biomarkers Prev. 2013 April; 22(4): 553-560. Most techniques relate to the detection of HPV-infections, which means that a cell that is capable of carcinogenesis is positive for human papilloma virus (HPV). It is also envisioned by the present invention that the cell, which is capable of carcinogenesis is a cell that is positive for (i) LGR6, LGR5 and/or LGR4; and (ii) human papilloma virus (HPV). Cells double-positive for both, LGR6, LGR5 and/or LGR4 and HPV can for example be reserve cells, cuboid cells or columnar cells. These cells have a high probability to transform into a HPV-induced precancerous lesion or a HPV-induced cancer. Thus, more than 1 %, 3 %, 5 %, 7 %, 10 %, 15 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 % 80 %, 90 %, 95 %, 99% or 100 % of the cells that are positive for (i) LGR6, LGR5 and/or LGR4; and (ii) human papilloma virus (HPV) will then result in a HPV-induced precancerous lesion and/or HPV-induced cancer.

[162] In addition to the detection of LGR6, LGR5 and/or LGR4 the methods of the present invention can also comprise the detection of HPV. HPV is a DNA virus from the papillomavirus family. HPVs can establish productive infections in keratinocytes of the skin or mucous membranes. Most HPV infections are subclinical and will cause no physical symptoms; however, in some subjects subclinical infections will become clinical and may cause benign papillomas (such as warts or squamous cell papilloma), or cancers of e.g. the cervix, vulva, vagina, penis, oropharynx and anus/anorectal region. Subclinical infections can also become clinical and may cause benign papillomas (such as warts or squamous cell papilloma), or cancers of e.g skin, head and neck (oropharynx, nasopharynx), oropharynx, esophagus, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder. In particular, HPV16 and HPV18 are known to cause around 60-80% of cervical cancer cases.

[163] Methods to detect HPV are known to the skilled artesian and for example described in Abreu et al. (2012) "A review of methods for detect human Papillomavirus infection." Virol J;9:262. HPV can for example be detected by the same means as LGR4, LGR5 and LGR6 as described herein. It is further envisioned by the present invention that HPV can be detected by DNA, RNA or protein analysis as described herein already for the detection of LGR6, LGR5 and/or LGR4. In particular, gene products of HPV can be detected. Such HPV specific gene products can be E1 , E2, E4, E5, E6, E7, L1 and/or L2. For example, detection of L1 and E4 can be used to detect a productive infection. Detection of E6 (Fa. Arbor vita) can for example be used to detect progressed lesions such as e.g. CIN2, CIN3/carcinoma in situ.

[164] HPV can also be detected via the detection of an upregulated cell cycle inhibitor. Exemplary cell cycle inhibitors include p14, p15, p16, p19, p21 or p27. For example, upreglation of p16 protein also termed p16^INK4a is a generally accepted biomarker of transforming HPV infections and precancerous lesions as also described in Ikenberg et al. (2013) "Screening for cervical cancer precursors with p16/Ki-67 dual-stained cytology: results of the PALMS study." J Natl Cancer lnst;105(20):1550-7. [165] The detection of an upregulated cell cycle inhibitor as described herein can also include additional detection of the absence or presence of a cell proliferation marker. Exemplary cell proliferation marker include Ki67, Ki-S5, Ki-S2, PCNA, POLD, BrdU, Mcm2, Mcm5PCNA, cyclins, helicases or subunits thereof, cell division cycle (cdc) molecules, phosphatase molecules, kinase molecules. Helicases or subunits thereof can for example include MCM2, MCM3, MCM4, MCM5, MCM6, MCM7 or HELAD1 . Exemplary cdc molecules, phosphatase molecules, kinase molecules can include CDC6, CDC7, protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 or MCM10. Suitable proliferation marker can for example include Ki-67, Ki-S2, Ki-S5, Mcm2, Mcm5PCNA, cyclins or BrdU.

[166] HPV can also be detected by the detection of both p16 and Ki-67 as for example described in Ikenberg et al. (2015) "Triaging HPV-positive women with normal cytology by p16/Ki-67 dual-stained cytology testing: Baseline and longitudinal data." Int J Cancer;136(10):2361 -8 or WO2004/038418.

[167] Additionally or alternatively HPV can also be detected via an epigenetic marker such as a marker for the detection of the methylation of HPV, cellular DNA, microRNA or immune scores. It can also be detected by cellular DNA via microRNAs or via immunoscores. Such immune scores are known to the skilled artesian and for example described in Galon et al. (2014) "Towards the introduction of the 'Immunoscore' in the classification of malignant tumours." J Pathol; 232: 199-209. In short, the immunoscore provides a score ranging from 0-4. An immunoscore of 0 indicates that low densities of two cell types, namely CD3/CD45RO, CD3/CD8 or CD8/CD45RO are found in the core of the tumor and the invasive margin of the tumor. An immunoscore of 4 indicates that high densities of both cell types are found in the core of the tumor and the invasive margin of the tumor.

[168] Additionally or alternatively HPV can also be detected via a cytological staining of said sample. In principle, the present invention encompasses any suitable cytological staining. Exemplary cytological stainings include DAPI, quinacrin, chromomycin, azan, acridin-orange, hematoxylin, eosin, sudan-red, toluidine-blue, papnicolaou or thioin.

[169] Such a cytological staining may further comprise a morphological evaluation of said sample. However, HPV may also solely be detected via morphological evaluation of said sample. In principle any suitable morphological method is encompassed by the present invention. Also such methods are known to the skilled artesian and for example described in Yamamoto (2004) "A morphological protocol and guide-list on uterine cervix cytology associated to papillomavirus infection." Rev. Inst. Med. trop. 46(4):189-193.

[170] The HPV to be detected may be any HPV. It is also envisioned by the present invention that the HPV to be detected is a high risk HPV or a low risk HPV. The low risk HPV are mostly associated with mild dysplasia and/or CIN1 lesions and rarely with malignancies. On the contrary, high risk HPV are strongly associated with precancerous lesions and cancer. Exemplary low risk HPVs include HPV 6, HPV 1 1 , HPV 40, HPV 42, HPV 43, HPV 44, HPV 54, HPV 61 , HPV 70, HPV 72 or HPV 81 . The high risk HPV can for example be a HPV16, HPV18, HPV31 , HPV33, HPV35, HPV39, HPV45, HPV51 , HPV52, HPV56, HPV58, HPV59 or HPV6. The high risk HPV can also further include HPV66, HPV73 or HPV82. Thus, the high risk HPV can for example be a HPV16, HPV18, HPV31 , HPV33, HPV35, HPV39, HPV45, HPV51 , HPV52, HPV56, HPV58, HPV59, HPV6, HPV66, HPV73 or HPV82. The HPV can also be a human HPV.

[171] The present invention also relates to a kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4. The detection of LGR6, LGR5 and/or LGR4 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. For example, the mean for the detection of LGR6, LGR5 and/or LGR4 can be binding protein as described herein. Examples of such a binding protein can include an anti- LGR4 antibody, an anti-LGR5 antibody and/or an anti-LGR6 antibody. The antibody can be a monoclonal or a polyclonal antibody. The antibody can also be a monoclonal antibody.

[172] The mean can also be a probe specific for LGR6, LGR5 and/or LGR4 such as an oligonucleotide as described herein.

[173] Additionally, the kit can further comprise a mean for the detection of HPV. The detection of HPV 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. For example, the mean for the detection of HPV can be a binding protein as described herein that specifically binds HPV, such as HPV protein. The mean for detecting HPV can also be an anti-p16 antibody and/or an anti-Ki-67 antibody. [174] In addition, the present invention relates to a use of LGR6, LGR5 and/or LGR4 for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV- induced cancer, wherein the use further comprises detecting human papilloma virus (HPV).

[175] Furthermore, the present invention relates to an in vitro method for detecting cells of the transformation zone of the cervix uteri and/or the anorectal region in a sample, the method comprising

(i) detecting LGR6, LGR5 and/or LGR4 in said sample, wherein the presence of LGR6, LGR5 and/or LGR4 indicates that the sample comprises cells of the transformation zone of the cervix uteri and/or the anorectal region, whereas the absence of LGR6, LGR5 and/or LGR4 indicates that the sample does not comprise cells of the transformation zone of the cervix uteri and/or the anorectal region.

[176] The present also relates to a use of LGR6, LGR5 and/or LGR4 for the reduction of false-negative results of samples obtained from cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. The present invention also relates to a use of LGR6, LGR5 and/or LGR4 for the reduction of false-negative results of samples obtained from cervix uteri and/or anorectal region.

[177] The present invention also relates to a use of LGR6, LGR5 and/or LGR4 for the detection of cells of a transition zone in a sample, wherein the transition zone is located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. The present invention also relates to a use LGR6, LGR5 and/or LGR4 for the detection of cells of the transformation zone of the cervix uteri and/or anorectal region in a sample.

[178] Notably, the methods and uses of the present invention as described herein can all be in vitro methods and uses.

[179] The present invention is further characterized by the following items:

[180] 1 . In vitro method for detecting cells of a transition zone in a sample, the method comprising

[181] 2. Method of item 1 , wherein the detection of LGR6, LGR5 and/or LGR4 is performed by DNA, RNA or protein analysis, preferably RNA or protein analysis.

[182] 3. Method of item 1 or 2, wherein the detection of the DNA is performed by PCR, southern blot, in situ hybridization or transcription-mediated amplification.

[183] 4. Method of any one of items 1 -3, wherein the detection of the RNA is performed by in situ hybridization, northern blot, RT-PCR or transcription-mediated amplification.

[184] 5. Method of any one of items 1 -4, wherein the detection of the protein is performed by immunohistochemistry, immunocytology, chromatographic methods or western blot.

[185] 6. Method of any one of items 1 -5, wherein the immunohistochemistry or immunocytology is a fluorescent or non-fluorescent immunohistochemistry or immunocytology.

[186] 7. 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.

[187] 8. Method of item 7, wherein the swab sample is a pap smear sample.

[188] 9. Method of item 7, wherein the tissue sample is a conisation sample.

[189] 10. Method of any one of items 1 -9, wherein the sample has been obtained from a subject.

[190] 1 1 . Method of item 10, wherein the subject is a vertebrate, preferably a human being.

[191] 12. Method of any one of items 1 -1 1 wherein the sample has been obtained from cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva. [192] 13. Method of any of items 1 -12, wherein the method is a method for the determination if a sample is suitable for HPV diagnosis.

[193] 14. Method of any of items 1 -13 for diagnosis and/or prognosis of a HPV- induced precancerous lesion or a HPV-induced cancer, wherein the method further comprises

(ii) detecting human papilloma virus (HPV) in said sample, wherein the detection of both LGR4, LGR5 and/or LGR6 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer.

[194] 15. Method of item 14, wherein the HPV-induced precancerous lesion comprises dysplasia.

[195] 16. Method of item 14 or 15, wherein the HPV-induced precancerous lesion is selected from the group consisting of high-grade squamous epithelial lesion, CIN2 or CIN3 (cervix uteri), high grade SIL (ASIL; anorectal region), VIN2, VIN3 (vulva), Sin2 or SIN3 (oropharynx/nasopharynx, oropharynx) or PelN2, PelN3 (penis).

[196] 17. Method of item 16, wherein the high-grade squamous epithelial lesion is a CIN3 lesion.

[197] 18. The method of item 14, wherein the HPV-induced precancerous lesion is a carcinoma in situ (CIS).

[198] 19. Method of item 18, wherein the CIS is an adenocarcinoma in situ, a squamous cell carcinoma in situ, a small cell carcinoma in situ, a neuroendocrine tumour in situ, a glassy cell carcinoma in situ or a villoglandular adenocarcinoma in situ, preferably an adenocarcinoma in situ or a squamous cell carcinoma in situ.

[199] 20. Method of item 14, wherein the method detects an adenocarcinoma in situ and a squamous cell carcinoma in situ.

[200] 21 . Method of item 14, wherein the cancer is selected from the group consisting of cancers of the cervix uteri, anus, penis, vulva nasopharynx/oropharynx or oropharynx such as tongue or tonsillar cancer.

[201] 22. Method of item 14, wherein the cancer is an invasive cancer.

[202] 23. Method of any one of items 14-22, wherein the prognosis comprises the detection of a cell, which is capable of carcinogenesis. [203] 24. Method of item 23, wherein the cell, which is capable of carcinogenesis is a reserve cell, a columnar cell or a cuboid cell.

[204] 25. Method of item 23 or 24, wherein the cell, which is capable of carcinogenesis is a cell that is positive for

(i) LGR4, LGR5 and/or LGR6; and

(ii) human papilloma virus (HPV).

[205] 26. Method of any one of items 14-25, wherein the detection of HPV is performed by DNA, RNA or protein analysis.

[206] 27. Method of item 26, wherein the HPV is detected via the detection of an upregulated cell cycle inhibitor, preferably the detection of an upregulated cell cycle inhibitor in the absence or presence of a cell proliferation marker.

[207] 28. Method of item 27, wherein the cell cycle inhibitor is selected from the group consisting of p14, p15, p16, p19, p21 or p27.

[208] 29. Method of item 27, wherein the cell proliferation marker is selected from the group consisting of Ki67, Ki-S5, Ki-S2, PCNA, POLD, BrdU, Mcm2, Mcm5PCNA, cyclins, helicases or subunits thereof, cell division cycle (cdc) molecules, phosphatase molecules, kinase molecules.

[209] 30. Method of item 29, wherein the helicases or subunits thereof are selected from the group consisting of MCM2, MCM3, MCM4, MCM5, MCM6, MCM7 or HELAD1 .

[210] 31 . Method of item 29, wherein the cdc molecules, phosphatase molecules, kinase molecules are selected from the group consisting of CDC6, CDC7, protein kinase, Dbf4, CDC14 protein phosphatase, CDC45 or MCM10.

[211] 32. Method of item 29, wherein the proliferation marker is selected from the group consisting of Ki-67, Ki-S2, Ki-S5, Mcm2, Mcm5PCNA, cyclins or BrdU.

[212] 33. Method of item 27, wherein the HPV is detected by p16 and Ki-67.

[213] 34. Method of any one of items 14-33, wherein the HPV is detected via an epigenetic marker such as a marker for the detection of the methylation of HPV or of cellular DNA, microRNA or immune scores. [214] 35. Method of any one of items 14-34, wherein the HPV is detected via a cytological staining of said sample, preferably further comprising a morphological evaluation of said sample.

[215] 36. Method of item 35, wherein the cytological staining is a staining with DAPI, quinacrin, chromomycin, azan, acridin-orange, hematoxylin, eosin, sudan-red, toluidine-blue or thioin.

[216] 37. Method of any one of items 14-36, wherein the HPV is a HPV16, HPV18, HPV31 , HPV33, HPV35, HPV39, HPV45, HPV51 , HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 or HPV82, preferably HPV16 and HPV18.

[217] 38. Method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample, wherein the detection of LGR4, LGR5 and/or LGR6 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV-induced cancer.

[218] 39. Method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample, wherein the detection of both LGR6, LGR5 and/or LGR4 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer and wherein the detection of LGR6, LGR5 and/or LGR4 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV-induced cancer.

[219] 40. Method of item 39, wherein the HPV-induced precancerous lesion comprises dysplasia.

[220] 41 . The method of item 39 or 40, wherein the HPV-induced precancerous lesion is a carcinoma in situ (CIS). [221] 42. Method of item 41 , wherein the CIS is an a squamous cell carcinoma in situ, an adenocarcinoma in situ, a small cell carcinoma in situ, a neuroendocrine tumour in situ, a glassy cell carcinoma in situ or a villoglandular adenocarcinoma in situ, preferably an adenocarcinoma in situ or a squamous cell carcinoma in situ.

[222] 43. Method of item 39-42, wherein the HPV-induced precancerous lesion is selected from the group consisting of high-grade squamous epithelial lesions such as CIN2 or CIN3 (cervix uteri), severe CIN (cornea/conjunctiva), high grade SIL (ASIL; anorectal region/anus), VIN2, VIN3 (vulva), SIN2, SIN3 (oropharynx/nasopharynx, oropharynx), VAIN2, VAIN3 (vagina) or PelN2, PelN3 (penis).

[223] 44. Method of item 43, wherein the high-grade squamous epithelial lesion is a CIN3 lesion.

[224] 45. Method of item 39, wherein the cancer is selected from the group consisting of cancers of the cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva.

[225] 46. Method of item 39 or 45, wherein the cancer is an invasive cancer.

[226] 47. Method of item 39, wherein the method detects an adenocarcinoma in situ and a squamous cell carcinoma in situ.

[227] 48. Kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4.

[228] 49. Kit of item 48, wherein the mean for the detection of LGR6, LGR5 and/or LGR4 is a binding protein, optionally the binding protein is an anti-LGR6 antibody, an anti-LGR5 antibody and/or an anti-LGR4 antibody.

[229] 50. Kit of item 48, wherein the mean for the detection of LGR6, LGR5 and/or LGR4 is a probe specific for LGR6, LGR5 and/or LGR4.

[230] 51 . Kit of any one of items 48-50, wherein the kit further comprises a mean for the detection of HPV.

[231] 52. Kit of item 51 , wherein the mean for the detection of HPV is an anti-p16 antibody and/or an anti-Ki-67 antibody.

[232] 53. Use of LGR6, LGR5 and/or LGR4 for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the use further comprises detecting human papilloma virus (HPV). [233] 54. Use of LGR6, LGR5 and/or LGR4 for the reduction of false-negative results of samples obtained from cervix uteri, anorectal region, oropharynx nasopharynx, oropharynx, penis or vulva.

[234] 55. Use of LGR6, LGR5 and/or LGR4 for the detection of cells of a transition zone in a sample, wherein the transition zone is located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.

[235] In the following table 1 different sequences are depicted.

S I Protein Sequence of (human) LGR4

MPGPLGLLCFLALGLLGSAGPSGAAPPLCAAPCSCDGDRRVDCSGKGLTAVPEGLSAFTQALDISMNNIT

E QLPEDAFKNFPFLEELQLAGNDLSFIHPKALSGLKELKVLTLQNNQLKTVPSEAIRGLSALQSLRLDANH ITSVPEDSFEGLVQLRHLWLDDNSLTEVPVHPLSNLPTLQALTLALNKISSIPDFAFTNLSSLWLHLHN Q NKIRSLSQHCFDGLDNLETLDLNYNNLGEFPQAIKALPSLKELGFHSNSISVIPDGAFDGNPLLRTIHLY ID DNPLSFVGNSAFHNLSDLHSLVIRGASMVQQFPNLTGTVHLESLTLTGTKISSIPNNLCQEQKMLRTLDL SYNNIRDLPSFNGCHALEEISLQRNQIYQIKEGTFQGLISLRILDLSRNLIHEIHSRAFATLGPITNLDV J SFNELTSFPTEGLNGLNQLKLVGNFKLKEALAAKDFVNLRSLSVPYAYQCCAFWGCDSYANLNTEDNSLQ DHSVAQEKGTADAANVTSTLENEEHSQI I IHCTPSTGAFKPCEYLLGSWMIRLTVWFIFLVALFFNLLVI

O: I LTTFASCTSLPSSKLFIGLISVSNLFMGIYTGILTFLDAVSWGRFAEFGIWWETGSGCKVAGFLAVFSSE SAIFLLMLATVERSLSAKDIMKNGKSNHLKQFRVAALLAFLGATVAGCFPLFHRGEYSASPLCLPFPTGE

1 I TPSLGFTVTLVLLNSLAFLLMAVIYTKLYCNLEKEDLSENSQSSMIKHVAWLIFTNCIFFCPVAFFSFAP LITAISISPEIMKSVTLIFFPLPACLNPVLYVFFNPKFKEDWKLLKRRVTKKSGSVSVSISSQGGCLEQD FYYDCGMYSHLQGNLTVCDCCESFLLTKPVSCKHLIKSHSCPALAVASCQRPEGYWSDCGTQSAHSDYAD EEDSFVSDSSDQVQACGRACFYQSRGFPLVRYAYNLPRVKD

Protein Sequence of (human) LGR5 isoform 2 precursor [Homo sapiens]

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSY

LDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSL

QSLRLDANHISYVPPSCFSGLHSLRHLWLDDNALTEIPVQAFRSLSALQAMTLALNKIHHIPDYAFGNLS

SLWLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKELHFYDNPIQFVGRSAFQHL

PELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLSYNLLEDLPSFSVCQ

KLQKIDLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFSTLPSLIKLDLSSNLLSSFPI GLHG

N LTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCCAFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQ

AQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLIRIGVWTIAVLALTCNALVTSTVFR

O: SPLYISPIKLLIGVIAAVNMLTGVSSAVLAGVDAFTFGSFARHGAWWENGVGCHVIGFLSIFASESSVFL 2 LTLAALERGFSVKYSAKFETKAPFSSLKVI ILLCALLALTMAAVPLLGGSKYGASPLCLPLPFGEPSTMG

YMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLLFTNCILNCPVAFLSFSSLINLT

FISPEVIKFILLVWPLPACLNPLLYILFNPHFKEDLVSLRKQTYVWTRSKHPSLMSINSDDVEKQSCDS

TQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSSVAFVPCL

Protein Sequence of (human) LGR5 isoform 3 precursor [Homo sapiens]

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSY

E LDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSL QSLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGNPS Q LITIHFYDNPIQFVGRSAFQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLP ID NLQVLDLSYNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFSTLP SLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCCAFGVCENAYKIS NQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLIRI GVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIAAVNMLTGVSSAVLAGVDAFTFGSFARHGAWWE NGVGCHVIGFLSIFASESSVFLLTLAALERGFSVKYSAKFETKAPFSSLKVI ILLCALLALTMAAVPLLG GSKYGASPLCLPLPFGEPSTMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLL FTNCILNCPVAFLSFSSLINLTFISPEVIKFILLVWPLPACLNPLLYILFNPHFKEDLVSLRKQTYVWT RSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSSVAFVPCL Protein Sequence of (human) LGR5 isoform 1 precursor [Homo sapiens]

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSY LDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSL

Q QSLRLDANHISYVPPSCFSGLHSLRHLWLDDNALTEIPVQAFRSLSALQAMTLALNKIHHIPDYAFGNLS ID SLWLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGN PSLITIHFYDNPIQFVGRSAFQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQ LPNLQVLDLSYNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCCAFGVCENAYK ISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLI RIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIAAVNMLTGVSSAVLAGVDAFTFGSFARHGAW

4 WENGVGCHVIGFLSIFASESSVFLLTLAALERGFSVKYSAKFETKAPFSSLKVI ILLCALLALTMAAVPL LGGSKYGASPLCLPLPFGEPSTMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIAL LLFTNCILNCPVAFLSFSSLINLTFISPEVIKFILLVWPLPACLNPLLYILFNPHFKEDLVSLRKQTYV WTRSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSSVAFVPCL

Protein Sequence of (human) LGR6 isoform 1 precursor [Homo sapiens]

MPSPPGLRALWLCAALCASRRAGGAPQPGPGPTACPAPCHCQEDGIMLSADCSELGLSAVPGDLDPLTAY LDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSGLYSLKILMLQNNQLGGIPAEALWELPSL

Q QSLRLDANLISLVPERSFEGLSSLRHLWLDDNALTEIPVRALNNLPALQAMTLALNRISHIPDYAFQNLT

|Q J SLWLHLHNNRIQHLGTHSFEGLHNLETLDLNYNKLQEFPVAIRTLGRLQELGFHNNNIKAIPEKAFMGN PLLQTIHFYDNPIQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQQ N I LPRLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRSIHPEAFST LHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILEVPYAYQCCPYGMCASFFK

O: I ASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQLEMEDSKPHPSVQCSPTPGPFKPCEYLFE SWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPVPLPPVKFWGAIAGANTLTGISCGLLASVDALTFGQFS

5 EYGARWETGLGCRATGFLAVLGSEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGLA AALPLASVGEYGASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLWAGAYIKLYCDLPRGDFEAVWDCA MVRHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLWLPLPACLNPLLYLLFNPHFRDDLRR LRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLETYGFPSVTLISCQQPGAP RLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSGGGGFQPSGLAFASHV

Protein Sequence of (human) LGR6 isoform 3 [Homo sapiens]

E MRLEGEGRSARAGQNLSRAGSARRGAPRDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSGL YSLKILMLQNNQLGGIPAEALWELPSLQSLDLNYNKLQEFPVAIRTLGRLQELGFHNNNIKAIPEKAFMG Q NPLLQTIHFYDNPIQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQ

|D J QLPRLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRSIHPEAFS TLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILEVPYAYQCCPYGMCASFF N I KASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQLEMEDSKPHPSVQCSPTPGPFKPCEYLF ESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPVPLPPVKFWGAIAGANTLTGISCGLLASVDALTFGQF

O: I SEYGARWETGLGCRATGFLAVLGSEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGL AAALPLASVGEYGASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLWAGAYIKLYCDLPRGDFEAVWDC

6 AMVRHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLWLPLPACLNPLLYLLFNPHFRDDLR RLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLETYGFPSVTLISCQQPGA PRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSGGGGFQPSGLAFASHV

Protein Sequence of (human) LGR6 isoform 2 [Homo sapiens]

MGRPRLTLVCQVSI I ISARDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSGLYSLKILMLQ NNQLGGIPAEALWELPSLQSLRLDANLISLVPERSFEGLSSLRHLWLDDNALTEIPVRALNNLPALQAMT

Q LALNRISHIPDYAFQNLTSLWLHLHNNRIQHLGTHSFEGLHNLETLDLNYNKLQEFPVAIRTLGRLQEL

|D I GFHNNNIKAIPEKAFMGNPLLQTIHFYDNPIQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEI LTLTRAGIRLLPSGMCQQLPRLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQA LDLSWNAIRSIHPEAFSTLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILE N VPYAYQCCPYGMCASFFKASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQLEMEDSKPHPS VQCSPTPGPFKPCEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPVPLPPVKFWGAIAGANTLTG

O: I SCGLLASVDALTFGQFSEYGARWETGLGCRATGFLAVLGSEASVLLLTLAAVQCSVSVSCVRAYGKSPS LGSVRAGVLGCLALAGLAAALPLASVGEYGASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLWAGAYI

7 KLYCDLPRGDFEAVWDCAMVRHVAWLI FADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLWLPLPACL NPLLYLLFNPHFRDDLRRLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLI LEASEAGRPPGLE TYGFPSVTLI SCQQPGAPRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSGGGGFQPSGLA FASHV

S EQ ID LGR4 DNA sequence [Homo sapiens]

h JO: 8

SEQ ID LGR5 DNA sequence [Homo sapiens]

NO: 9

SEQ ID LGR6 DNA sequence [Homo sapiens]

NO: 10

SEQ ID LGR4 mRNA [Homo sapiens]

NO: 1 1

SEQ ID LGR5 mRNA, variant 2 [Homo sapiens]

NO: 12

SEQ ID LGR5 transcript variant 3, mRNA [Homo sapiens]

NO: 13

SEQ ID LGR5 transcript variant 1 , mRNA [Homo sapiens]

NO: 14

SEQ ID LGR6 transcript variant 1 , mRNA [Homo sapiens]

NO: 15

SEQ ID LGR6 transcript variant 3, mRNA [Homo sapiens]

NO: 16

SEQ ID LGR6 transcript variant 2, mRNA [Homo sapiens]

NO: 17

Table 1 . Sequences as described herein. [236] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

EXAMPLES

[237] EXAMPLE 1

[238] Paraffin sections were cut into 2 μιτι sections. These were deparaffinized with xylene (3 x 10 min) and ethanol in decreasing concentrations (2 min each) and rehydrated for 5 min in A. dest.. The unmasking of antigens was done with sodium- citrate buffer pH 7.0 (as mentioned above) in a microwave reaching boiling temperature several times for 10 min and then cooled down to room temperature (RT) for at least 15 min. Immunohistochemical staining was done according to the manufacturer^'s instructions (Vector Laboratories, Burlingame, USA) with minor modifications.

[239] In case of the Peroxidase-staining (mouse primary antibodies), the ImmPRESS Anti-Mouse Ig (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, USA) was used and endogenous peroxidases were blocked with 3% H2O2 in 1 xTBS pH 7.6 for 10 min in a light-protected manner.

[240] For rabbit primary antibodies, the ImmPRESS-AP Anti-Rabbit Ig (alkaline phosphatase) Polymer Detection Kit (Vector Laboratories, Burlingame, USA) was used.

[241] In all cases, slides were washed for 5 min in 1 xTBS pH 7.6. Then 2.5% normal horse serum was added for 20 min at RT. Primary antibodies were diluted in 0.5% BSA (bovine serum albumin) in 1 xTBS pH 7.6 at the dilutions indicated in Table 1 and the slides were incubated overnight at 4°C in a moisture chamber. After 5 min washing in 1 xTBS pH 7.6, the respective secondary enzyme-coup led antibody solution was added for 30 min. After 2x washing in 1 xTBS pH 7.6 for 5 min each, the substrate was diluted as indicated in the manufacturer^'s protocol and sections were incubated in a light-protected manner for the time intervals indicated in Table 1 . After washing with tab water for 2 min, sections were counterstained with hematoxylin for 20-40 s and the slides were washed for 3-5 min with warm tab water. After dehydration with increasing ethanol concentrations, slides were shortly incubated in xylene and embedded with mounting solution.

[242] For double-staining, the Peroxidase-staining protocol was done first using the ImmPRESS Anti-Mouse Ig (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, USA). After adding the Peroxidase-substrate and washing with 1 xTBS buffer pH 7.6 for 5 min, the staining was continued with 2.5% normal horse serum as described above using the ImmPRESS-AP Anti-Rabbit Ig (alkaline phosphatase) Polymer Detection Kit (Vector Laboratories, Burlingame, USA).

The antibodies used include:

monoclon

al p63 Mouse lgG2a 4A4 1 :1500 Santa Cruz 10

monoclon

al

Table 2. Antibodies used in the Examples.

[243] The results of the different stainings are shown in the Figures. In Figure 1 the transformation zone (TZ) of a normal cervix uteri in comparison to the ectocervical tissue is shown. A and B were stained with anti-LGR4 Ab (1 :300), C-E with anti- LGR5 Ab (1 :300-1 :400), F-l with anti-LGR6 Ab (1 :400), J with anti-CD63 Ab (1 :2000), K-N were double-stained with anti-LGR6 Ab (1 :500) and anti-p63 (1 :1500). In Figure 1 (A), (C), (D), (F), (H), (I), (J) and (K) the TZ also comprises a metaplasia. While in Figure 1 (B), (E) and (G) a normal TZ without metaplasia is shown. Stainings show that LGR4 (A), 5 (C, D) and 6 (F, H, I) are expressed in subcolumnar and subglandular reserve cells, immature metaplasia, in some but not all columnar cells within the TZ and that LGR4 (B), 5 (E) and 6 (G) are not expressed in normal ectocervix. To further confirm that LGR4, LGR5 and LGR6 expression is present in reserve cells a staining with p63 was conducted. p63 is expressed reserve cells but also in basal cell of squamous epithelium and squamous cell carcinomas. Basal cells of subcolumnar immature metaplasia stain double-positive for p63 and LGR6 (K). Subglandular reserve cells stain double-positive for LGR6 and p63 (L-N). LGR4, LGR5 and LGR6 are furthermore expressed in subcolumnar and subglandular reserve cells, immature metaplasia, in some but not all columnar cells within the TZ and that LGR4, 5 and 6 are not expressed in normal ectocervix.

[244] Figure 2 shows the extension of the TZ into the endocervical canal of a normal cervix uteri, which does not comprise a metaplasia. The TZ was stained with anti- LGR4 Ab (1 :200), B with anti-LGR5 Ab (1 :300), C with anti-LGR6 Ab (1 :400), D with anti-CD63 Ab (1 :2000). Stainings show that LGR4, 5, and 6 expression is strongest in columnar cells close to the squamocolumnar junction of the TZ and expression strongly decreases towards the endocervical canal (A-C). CD63 in contrast is expressed in all endocervical columnar cells at equal intensity (D).

[245] In Figures 3 to 5 precancerous lesions such as high-grade squamous intraepithelial lesion (HSIL) of the cervix uteri are shown. Importantly, the sections shown in each of Fig. 3-5 (A)-(F) represent subsequent sections. Therefore, these sections also allow for the evaluation, if the lesion also stains for different marker such as Ki67 and LGR4, 5 or 6 or Ki67 and CD63. (A) was stained with anti-LGR4 Ab (1 :350), (B) with anti-LGR5 Ab (1 :300-1 :500), (C) with anti-LGR6 Ab (1 :300-400), (D) with anti-p16 (1 :32-64), € with anti-Ki-67 (1 :1200) and (F) with anti-CD63 Ab (1 :2000). Staining for p16 was performed to detect HPV infections. Often additionally a Ki-67 staining was performed. A cell positive for both Ki-67 andp16 will be infected with HPV. Furthermore, the presence of both markers in some cells detects the presence of a precancerous lesion such as HSIL.

[246] Stainings show that p16- and Ki-67-positive epithelial cells of the precancerous lesion (HSIL) express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in gland cells and in only some cell layers located above the LGR4-, 5-, 6-, p16- and Ki-67-positive HSIL. The majority of CD63-positive cells are however p16 low to negative and Ki-67 negative. This shows that precancerous lesions originate from LGR4, LGR 5 or LGR 6 positive cells but not from CD63- positive cells.

[247] In Figure 6 a squamous cell carcinoma next to a precancerous lesion such as HSIL of the cervix uteri is shown. (A) was stained with anti-LGR4 Ab (1 :350), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64),€ with anti-Ki-67 (1 :1200) and (F) with anti-CD63 Ab (1 :2000).

[248] Stainings show that p16- and Ki-67- positive epithelial cells of the squamous cell carcinoma and precancerous lesion (HSIL) express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in cells located above the LGR4-, 5-, 6-, p16- and Ki-67-positive precancerous lesion (HSIL). The majority of CD63- positive cells are p16 low to negative and Ki-67-negative.

[249] In Figure 7 a squamous cell carcinoma of the cervix uteri is shown. (A) was stained with anti-LGR4 Ab (1 :200), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), (E) with anti-Ki-67 (1 :1200) and (F) with anti- CD63 Ab (1 :2000).

[250] These further stainings confirm that p16- and Ki-67-positive epithelial cells of the squamous cell carcinoma express LGR4, 5, and 6 but not CD63. In contrast, CD63 expression is detected in cells located beneath the LGR4-, 5-, 6-, p16- and Ki- 67-positive squamous cell carcinoma. The majority of CD63-positive cells are p16- negative and Ki-67-negative. Thus, LGR4, 5, and 6 can also be used to detect squamous cell carcinoma, while CD63 will not detect this type of cancer.

[251] In Figures 8 and 9 adenocarcinomas of the cervix uteri are shown. (A) was stained with anti-LGR4 Ab (1 :200), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64),€ with anti-Ki-67 (1 :1200) and (F) with anti-CD63 Ab (1 :2000).

[252] Stainings show that p16- and Ki-67-positive epithelial cells of the adenocarcinomas express LGR4, 5, and 6 and low levels of CD63. Thus, LGR4, 5, and 6 can also be used to detect adenocarcinomas.

[253] In Figure 10 a CIN3 lesion in subsequent slices of the cervix uteri is shown. Staining with anti-LGR4 Ab (1 :200), with anti-LGR6 Ab (1 :400), (D) with anti-p16 (1 :64), with anti-CD63 Ab (1 :2000) was performed. Stainings show that p16-positive epithelial cells of the CIN3 lesion expresses LGR4, and 6 and p16. However, CD63 is not detected in this lesion. Thus, LGR4, 5, and 6 can also be used to detect CIN3 lesions.

[254] EXAMPLE 2

[255] Methods

[256] Immunohistochemistry

[257] Sections of anorectal normal tissue and perianal carcinoma in situ were stained as described for cervical tissue.

[258] Immunocytochemistry

[259] The Hologic semi-automated sample processor was used to load SuperFrost Plus® slides (R. Langenbrinck, Emmendingen, Germany) with ThinPrep® cervical samples. After drying the slides overnight at room temperature they were incubated with preheated citrate buffer at 95°C for 5 min, washed twice with PBS, incubated in 3% H2O2 in PBS for 10 min to block endogenous peroxidases and washed again twice. After blocking with 2.5% normal horse serum (Vector Laboratories, Eching, Germany) for 1 h at room temperature, samples were incubated with primary antibodies (listed in Table 3) diluted in 0.5% BSA in PBS for either 2 h at room temperature or overnight at 4°C and washed twice with PBS for 10 min. Slides were then incubated with secondary antibody ImmPRESS™ Anti-Rabbit IgG Peroxidase (Vector Laboratories) for 30 min at room temperature, washed twice with PBS for 5 min and developed for approximately 5 min with ImmPACT DAB Peroxidase Substrate (Vector Laboratories), diluted according to the manufacturer's protocol (1 drop/ml diluent). Slides were carefully rinsed twice with A. dest. and washed with tab water before staining with haematoxylin for 40 sec. Then they were again rinsed and incubated in tab water for 5 min, dehydrated in ethanol solutions with increasing concentrations (50% ->70% ->80% ->90%, with gentle shaking 5-6x times at each step followed by 99% ethanol for 2 min) and incubated with xylol for 2-3 sec. Slides were air dried and covered with a drop of VectaMount Permanent Mounting Medium (Vector Laboratories) and a cover slip.

[260] Table 3: Primary antibodies for immunocytochemistry

Antibody Clonality Species Dilution Manufacturer

LGR4 polyclonal rabbit-IgG 1 :300 Sigma Aldrich,

(GPCR GPR48) Steinheim

LGR5 polyclonal rabbit-IgG 1 :300 Abeam,

(GPCR GPR49) Cambridge

LGR6 polyclonal rabbit-IgG 1 :300 Abeam,

Cambridge

[261] Results

[262] Sections of anorectal normal tissue and perianal carcinoma in situ were stained as described for cervical tissue (Fig. 1 1 ). Shown in Figure 1 1 is the normal anorectal transition zone (or transformation zone; TZ) (A) of a patient in comparison to anorectal glandular tissue outside the transformation zone (B) or normal anal mucosa outside the transformation zone (C). (A), (B) and were stained with anti- LGR6 Ab (1 :400). Stainings show that LGR6 expression is strongest in epithelial cells close to the squamocolumnar junction of the TZ and in metaplastic cells. Anorectal glands or stratified epithelium outside the transformation zone are negative. Therefore, transformation zone of the anorectal region stains positive for LGR6. From these results it can further be obtained that the transformation zone of the anorectal region will also stain positive for LGR4 and LGR5, as shown e.g. for the cervix uteri in Example 1 . Here, the transformation zone also stained positive for LGR4, LGR5 as well as LGR6.

[263] In Figure 12 precancerous lesions such as anal high-grade squamous intraepithelial lesion (AIN3, anal carcinoma in situ) from two different patients (A) and (B) stained with anti-LGR6 Ab (1 :400) are shown. In Fig. 12 (B) it is shown that epithelial cells adjacent to the AIN3 also stain positive for LGR6. Thus, anal carcinoma in situ stains positive for LGR6. From these results it can be obtained that anal carcinoma stains positive for LGR6. This indicates that anal carcinoma will also stain positive for LGR4 and LGR5, as shown for the cervix uteri in Example 1 .

[264] Normal human foreskin was stained as described for cervical tissue with anti- LGR6 Ab (1 :400). The results obtained by this staining are shown in Figure 13. Stainings show that LGR6 expression is detected in stratifying epithelium. This means that epithelial cells in the transition zone in the penis is also stainable using LGR6, and presumably also by staining with LGR5 and/or LGR4.

[265] Immunocytochemical stainings of cervical ThinPreps samples from different patients with normal Pap smears are depicted in Figure 14. Samples were stained with anti-LGR4 Ab (1 :300), (B) with anti-LGR5 Ab (1 :300), (C) with anti-LGR6 Ab (1 :300). Brown color indicates positive staining. Stainings show a LGR4- (A), LGR5- (B) or LGR6- (C) -positive epithelial cell indicating that the swabs were taken from the TZ. This experiment thus provides ample evidence that LGR4, LGR5 and/or LGR6 can be stained in samples obtained from patients such as Pap smears.

[266] It must be 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.

[267] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[268] 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.

[269] 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 sometimes when used herein with the term "having".

[270] When used herein "consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[271] For each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms.

[272] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, 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.

[273] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as "(from) X to Y" equates with an interval which is defined as "between X and Y". Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of "5 mg/ml to 10 mg/ml" or "between 5 mg/ml and 10 mg/ml" includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value. REFERENCES

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Claims

1 . In vitro method for detecting cells of a transition zone in a sample, the method comprising

2. Method of claim 1 , wherein the detection of LGR6, LGR5 and/or LGR4 is performed by DNA, RNA or protein analysis, preferably RNA or protein analysis.

3. Method of claim 1 or 2, wherein the sample is a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine.

4. Method of claim 3, wherein the swab sample is a pap smear sample.

5. Method of claim 3, wherein the tissue sample is a conisation sample.

6. Method of any one of claims 1 -5, wherein the sample has been obtained from a subject.

7. Method of any of claims 1 -6, wherein the method is a method for the determination if a sample is suitable for HPV diagnosis.

8. Method of any of claims 1 -7 for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method further comprises

(ii) detecting human papilloma virus (HPV) in said sample, wherein the detection of both LGR6, LGR5 and/or LGR4 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer.

9. Method of claim 8, wherein the HPV-induced precancerous lesion comprises dysplasia.

10. Method of claim 8 or 9, wherein the HPV-induced precancerous lesion is selected from the group consisting of a high-grade squamous epithelial lesion, CIN2+ or CIN3 (cervix uteri), high grade SIL (ASIL; anorectal region), VIN2 or VIN3 (vulva) or SIN2 or SIN3 (oropharynx/nasopharynx, oropharynx) or PelN2, PelN3 (penis).

1 1 . Method of claim 10, wherein the high-grade squamous epithelial lesion (HSIL) is a CIN3 lesion.

12. The method of claim 10 or 1 1 , wherein the HPV-induced precancerous lesion is a carcinoma in situ (CIS).

13. Method of claim 12, wherein the CIS is an adenocarcinoma in situ, a squamous cell carcinoma in situ, a small cell carcinoma in situ, a neuroendocrine tumour in situ, a glassy cell carcinoma in situ or a villoglandular adenocarcinoma in situ, preferably a squamous cell carcinoma in situ or an adenocarcinoma in situ.

14. Method of claim 8, wherein the method detects an adenocarcinoma in situ and a squamous cell carcinoma in situ.

15. Method of claim 8, wherein the cancer is selected from the group consisting of cancers of the cervix uteri, anus, penis, vulva, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer.

16. Method of claim 8 or 15, wherein the cancer is an invasive cancer.

17. Method of any one of claims 8-16, wherein the prognosis comprises the detection of a cell, which is capable of carcinogenesis.

18. Method of claim 17, wherein the cell, which is capable of carcinogenesis is a reserve cell, a columnar cell or a cuboid cell.

19. Method of claim 17 or 18, wherein the cell, which is capable of carcinogenesis is a cell that is positive for

(i) LGR6, LGR5 and/or LGR4; and

(ii) human papilloma virus (HPV).

20. Method of any one of claims 8-19, wherein the detection of HPV is performed by DNA, RNA or protein analysis.

21 Method of claim 20, wherein the HPV is detected via the detection of an upregulated cell cycle inhibitor, preferably the detection of an upregulated cell cycle inhibitor in the absence or presence of a cell proliferation marker.

22. Method of any one of claims 8-21 , wherein the HPV is a HPV16, HPV18, HPV31 , HPV33, HPV35, HPV39, HPV45, HPV51 , HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 or HPV82, preferably HPV16 and HPV18.

23. Method of any of claims 8-22, wherein the detection of LGR4, LGR5 and/or LGR6 and no HPV in the sample is indicative of the absence of a HPV- induced precancerous lesion or a HPV-induced cancer.

24. Method for diagnosis and/or prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer, wherein the method comprises

(i) detecting LGR6, LGR5 and/or LGR4; and

(ii) detecting human papilloma virus (HPV) in a sample, wherein the detection of both LGR6, LGR5 and/or LGR4 and HPV in the sample is indicative of HPV-induced precancerous lesion or HPV-induced cancer and wherein the detection of LGR6, LGR5 and/or LGR4 and no HPV in the sample is indicative of the absence of a HPV-induced precancerous lesion or a HPV- induced cancer.

25. Method of claim 24, wherein the HPV-induced precancerous lesion is selected from the group consisting of high-grade squamous epithelial lesion, CIN2 or CIN3 (cervix uteri), severe CIN (cornea/conjunctiva), high grade SIL (ASIL; anorectal region/anus), VIN2, VIN3 (vulva), SIN2, SIN3 (oropharynx/nasopharynx, oropharynx), VAIN2, VAIN3 (vagina), PelN3 or PelN3 (penis).

26. Method of claim 24, wherein the cancer is selected from the group consisting of cancers of the cervix uteri, skin, anus, vagina, vulva, penis, oropharynx/nasopharynx, oropharynx such as tongue or tonsillar cancer or cornea/conjunctiva. Method of claim 24 or 26, wherein the cancer is an invasive cancer.

Kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4.

Kit of claim 28, wherein the kit further comprises a mean for the detection of HPV.

Use of LGR6, LGR5 and/or LGR4 for diagnosis and/or prognosis of a HPV- induced precancerous lesion or a HPV-induced cancer, wherein the use further comprises detecting human papilloma virus (HPV).

Use of LGR6, LGR5 and/or LGR4 for the reduction of false-negative results of samples obtained from cervix uteri, anorectal region, nasopharynx/oropharynx, oropharynx, penis or vulva.

Use of LGR6, LGR5 and/or LGR4 for the detection of cells of a transition zone in a sample, wherein the transition zone is located in the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.