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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/708—Specific hybridization probes for papilloma
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• 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).
• 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).
• HPV infections e.g. genital HPV infection
• about two thirds of the HPV infections 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.
• transition zone- where squamous and columnar epithelium meet.
• transition zones also called transformation zones
• 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.
• 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.
• 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
• 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
• the present invention relates to a kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4.
• the kit further comprises a mean for the detection of HPV.
• 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).
• HPV human papilloma virus
• the present invention relates to an in vitro method for detecting cells of a transition zone in a sample, the method comprising
• 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.
• 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.
• 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.
• SCJ current squamocolumnar junction
• 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.
• 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 LGR4, 5 and 6 are not expressed in normal ectocervix.
• FIG. 1 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).
• FIG. 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.
• HSIL high-grade squamous intraepithelial lesion
• Stainings show that p16- and Ki-67-positive epithelial cells of the precancerous lesion (HSIL) express LGR4, 5, and 6 but not CD63.
• HSIL precancerous lesion
• 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.
• FIG. 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).
• 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.
• 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.
• FIG. 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).
• FIGs 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).
• 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.
• FIG. 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.
• FIG. 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.
• AIN3 anal high-grade squamous intraepithelial lesion
• 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.
• HPV-induced carcinogenesis e.g. cervical carcinogenesis
• HPV infection e.g. HIV infection
• persistence rather than clearance of the virus
• 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).
• 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.
• HPV testing as a primary test with secondary triage testing or HPV co-testing has been introduced into screening programs in different countries.
• 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 ).
• HPV testing cannot discriminate between transient and persistent infection, a prerequisite for cancer development.
• 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.
• 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.
• stem cell markers LGR4, 5 and 6 characterizes cells from which HPV-induced precancerous lesions and HPV-induced cancer such as cervical cancer originate.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• the method of the present invention has important consequences for cancer and precancerous lesion screening, prognosis and diagnosis:
• 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.
• 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.
• 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.
• LGR4, LGR5, and/or LGR6-staining of conisation specimens will provide information whether the susceptible cells within the transition zone have been removed.
• investigations using the LGR4, 5 and/or 6 are also suitable to exclude relevant HPV-induced precancerous lesions such as dysplasia, and cancers.
• the present invention relates to an in vitro method for detecting cells of a transition zone in a sample, the method comprising
• 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.
• a transition zone 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.
• 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.
• 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.
• Stratified squamous epithelia may be either keratinized or non-keratinized.
• 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.
• 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 basement of connective tissue. This is for example the case for the transition zone of the vulva or penis as 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).
• 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.
• HPV-induced cancer and/or HPV-induced precancerous lesion may also arise from any transition zone as described herein.
• 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.
• HPV human papilloma virus
• 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.
• 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.
• 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.
• the transition zone can for example be located in the penis.
• This transition zone can be e.g. located in the foreskin.
• 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.
• the transitional zone of the penis can comprise mucosal stratifying epithelium.
• 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.
• Cells of the transition zones as described herein can be detected by the detection of LGR6, LGR5 and/or LGR4.
• LGR4 GPR48
• LGR4 embraces any LGR4 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
• 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 (
• LGR5 (GPR49, GPR67) is also termed "Leucine-rich repeat-containing G- protein coupled receptor 5".
• LGR5 embraces any LGR5 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
• 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.
• LGR6 is also termed "Leucine-rich repeat-containing G-protein coupled receptor 6", respectively.
• LGR6 embraces any LGR6 nucleic acid molecule or polypeptide and can also comprise fragments or variants thereof.
• 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.
• 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.
• 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
• a "variant" of the polypeptide of the present invention encompasses a polypeptide wherein one or more amino acid residues are substituted.
• 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.
• 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.
• 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.
• 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.
• BLAST and BLAST 2.0 algorithms are also available to those having skill in this art.
• 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.
• the BLASTP program uses as defaults a word size (W) of 3, and an expectation (E) of 10.
• the BLOSUM62 scoring matrix Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) can be used.
• 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.
• 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.
• 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.
• 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 .
• DNA or methylation pattern or histone modifications of the DNA
• 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
• 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 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.
• 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.
• oligonucleotides also called primers
• LGR6, LGR5 and/or LGR4 or fragments or variants thereof or HPV DNA or RNA such as mRNA 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• immunohistochemistry and immunocytology can comprise the use of antibodies to detect the LGR6, LGR5 and/or LGR4 protein.
• both immunohistochemistry and immunocytology can also comprise fluorescent or non-fluorescent immunohistochemistry and/or immunocytology.
• 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.).
• Ig's immunoglobulins of different classes
• IgA, IgG, IgM, IgD and IgE immunoglobulins
• subclasses such as lgG1 , lgG2 etc.
• 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).
• 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).
• the binding protein can also be an "antibody fragment".
• 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).
• the binding protein may also only have a single binding site, i.e., may be monovalent.
• monovalent binding proteins include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties.
• 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.
• the antibody or antibody fragment may be monoclonal or polyclonal.
• 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.
• the sample can be a swab, a brush, a liquid cytology specimen, a self-sample, a lavage, a biopsy, a tissue sample or urine.
• An exemplary swab sample can be a pap (papanicolau) smear sample.
• pap papanicolau
• 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.
• a sample, such as a pap smear sample can be obtained from the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.
• the sample such as the pap smear sample can be obtained from the cervix uteri or anorectal region.
• 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.
• 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.
• the sample has been obtained from a subject.
• exemplary sites from which a sample can be obtained include cervix uteri, anorectal region, vagina, vulva, penis, oropharynx such as tongue or tonsil, conjunctiva/limbus or skin.
• the sample can also be obtained from the cervix uteri, anorectal region, oropharynx/nasopharynx, oropharynx, penis or vulva.
• the sample can also be obtained from the cervix uteri or anorectal region.
• the 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.
• a mammal is as a human, dog, cat, cow, pig, mouse, rat etc.
• the mammal can also be a human being.
• the subject can be a vertebrate, preferably a human being.
• 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
• 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.
• 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
• 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.
• the HPV can be a high risk HPV.
• 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.
• 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.
• the presence of LGR6, LGR5 and/or LGR4 and HPV is detected at the same time in a sample.
• 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.
• 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.
• 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.
• 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.
• 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.
• marker of interest such as LGR6, LGR5 and/or LGR4, HPV or a cell cycle inhibitor
• the methods of the present invention can also be used for the prognosis of a HPV-induced precancerous lesion or a HPV-induced cancer.
• 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.
• 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).
• HPV-induced precancerous lesion and a HPV-induced cancer are 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.
• HPV-induced carcinogenesis e.g. cervical carcinogenesis
• HPV human papillomavirus
• HPV 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)
• a HPV- induced precancerous lesion or HPV-induced cancer as described herein can be prognosed.
• 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.
• HPV-induced precancerous lesions and/or HPV-induced cancers of the cervix uteri, vulva, anus, nasopharynx/oropharynx and oropharynx can be prognosed by the methods of the present invention.
• HPV-induced precancerous lesions and/or HPV-induced cancers of skin, head and neck 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.
• 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.
• cervical cancers such as e.g. carcinoma
• 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.
• 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.
• 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.
• 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.
• 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.
• HPV-induced precancerous lesion can be associated with high risk HPVs as described herein.
• the HPV- induced precancerous lesion can be associated with HPV 16, HPV 18, HPV 31 and/or HPV 33.
• 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.
• dysplasia may be detected via microscopically visible changes in cell morphology.
• Dysplasia of cells can be recognized in a pap smear or in another sample as described herein.
• 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.
• 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.
• 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.
• 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 %.
• the low grade HPV-induced precancerous lesions may also be a low grade squamous intraepithelial lesion (LSIL).
• 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.
• 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.
• 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.
• CIN2 moderate dysplasia
• CIN2 severe dysplasia
• CIN3 carcinoma in situ
• the high grade squamous epithelial lesions may be a CIN3 or CIN2 lesion, wherein the CIN2 lesion can be positive for p16.
• CIN2+ when used herein means a precancerous lesion that is CIN2 or CIN3.
• 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.
• CIN precancerous lesions
• the HPV-induced precancerous lesion can also be a conjuntival intraepithelial neoplasia (CIN).
• CIN conjuntival intraepithelial neoplasia
• 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.
• 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.
• HPV-induced precancerous lesion can also be an anal squamous intraepithelial lesion (ASIL), sometimes also referred to as AIN.
• ASIL anal squamous intraepithelial lesion
• AIN anal squamous intraepithelial lesion
• 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.
• 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).
• SCC squamous cell carcinoma
• 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.
• the precancerous lesion can also be a vaginal intraepithelial neoplasia (VAIN).
• VAIN vaginal intraepithelial neoplasia
• 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).
• Pap smear Papanicolaou test
• VAIN comes in three stages, VAIN1 , 2, and 3.
• 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.
• 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.
• SILs squamous intraepithelial lesions
• 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).
• CIN2+ or CIN3 cervix uteri
• severe CIN cornea/conjunctiva
• high grade SIL ASIL; anorectal region/anus
• VIN2/3 vulva
• SIN2 or SIN3 oropharynx
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• HPV human papilloma virus
• 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.
• benign papillomas such as warts or squamous cell papilloma
• cancers 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.
• benign papillomas such as warts or squamous cell papilloma
• cancers e.g skin, head and neck (oropharynx, nasopharynx), oropharynx, esophagus, lung, penis, vagina, prostate, cervix, anorectal region/anus, and bladder.
• HPV16 and HPV18 are known to cause around 60-80% of cervical cancer cases.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• epigenetic marker such as a marker for the detection of the methylation of HPV, cellular DNA, microRNA or immune scores.
• cellular DNA can also be detected by cellular DNA via microRNAs or via immunoscores.
• 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.
• 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.
• HPV can also be detected via a cytological staining of said sample.
• 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.
• Such a cytological staining may further comprise a morphological evaluation of said sample.
• HPV may also solely be detected via morphological evaluation of said sample.
• 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.
• 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.
• 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.
• 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.
• 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.
• the mean can also be a probe specific for LGR6, LGR5 and/or LGR4 such as an oligonucleotide as described herein.
• 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.
• 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.
• 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).
• HPV human papilloma virus
• 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
• 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.
• 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.
• the present invention is further characterized by the following items:
• 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).
• CIN2 or CIN3 cervix uteri
• ASIL anorectal region
• VIN2, VIN3 vulva
• Sin2 or SIN3 oropharynx/nasopharynx, oropharynx
• PelN2 PelN3
• 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.
• 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.
• 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.
• 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.
• cytological staining is a staining with DAPI, quinacrin, chromomycin, azan, acridin-orange, hematoxylin, eosin, sudan-red, toluidine-blue or thioin.
• HPV is a HPV16, HPV18, HPV31 , HPV33, HPV35, HPV39, HPV45, HPV51 , HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 or HPV82, preferably HPV16 and HPV18.
• CIS carcinoma in situ
• the HPV-induced precancerous lesion is a carcinoma in situ (CIS).
• 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.
• 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).
• 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, Pel
• cancer 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.
• Kit comprising a mean for the detection of LGR6, LGR5 and/or LGR4.
• 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.
• Kit of item 51 wherein the mean for the detection of HPV is an anti-p16 antibody and/or an anti-Ki-67 antibody.
• 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).
• HPV human papilloma virus
• SEQ ID LGR5 transcript variant 3 mRNA [Homo sapiens]
• SEQ ID LGR5 transcript variant 1 mRNA [Homo sapiens]
• SEQ ID LGR6 transcript variant 1 mRNA [Homo sapiens]
• SEQ ID LGR6 transcript variant 3 mRNA [Homo sapiens]
• SEQ ID LGR6 transcript variant 2 mRNA [Homo sapiens]
• FIG. 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).
• FIG. 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).
• 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.
• 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.
• 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.
• FIG. 1 1 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.
• 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.
• 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.
• 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.
• 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.
• LSIL-H Low-grade squamous intraepithelial lesion/cannot exclude high-grade squamous intraepithelial lesion
• Martens JE Smedts FM, Ploeger D, Helmerhorst TJ, Ramaekers FC, Arends JW, et al. Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal.
• Martens Smedts, van Muyden, Ruud, Schoots, Helmerhorst, Hopman, Ramaekers, Arends Reserve Cells in Human Uterine Cervical Epithelium Are Derived from Mullerian Epithelium at Midgestational Age International Journal of Gynecological Pathology October 2007 - Volume 26 - Issue 4 - pp 463-468

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