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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • 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
• • 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
  • G01N33/57407—Specifically defined cancers
  • G01N33/57411—Specifically defined cancers of cervix
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—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

• HPVs Human Papillomaviruses
• SUMO Small Ubiquitin-like Modifier
• the present invention refers to a bio marker consisting in a protein involved in the sumoylation pathway or an mRNA encoding the same for use in assessing the risk and/or diagnosing and/or prognosing and/or monitoring the progression of an HPV oncogenic infection, to an in vitro method for assessing the risk and/or diagnosing and/or prognosing and/or monitoring the progression of an HPV oncogenic infection and to a diagnostic kit for HPV oncogenic infection.
• HPVs Human papillomaviruses
• HPVs can be classified as low-risk and high-risk viruses: low-risk HPVs are associated with a spectrum of benign warts, such as condyloma [22], whereas infections with high-risk HPVs, such as HPV 16 (European German, GenBank Accession number AF536179) and 18 (Human papillomavirus type 18, GenBank Accession number X05015.1), are manifested by intraepithelial malignant progression caused by viral E6 and E7 [1].
• High-risk E6 and E7 are considered as oncogenes that, through the degradation of p53 and Rb by ubiquitin-proteasome pathway, respectively, cause perturbation of cell cycle regulation in the infected cell [1].
• the strategy is to strongly aim and focus on persistent infection caused by HPV. Besides laboratory tests to detect HPV DNA, there are other promising markers of HPV oncogenic infection. These include: i) markers of increased HPV mR A and protein expression; ii) markers of increased cell proliferation, such as Ki-67, MCM2, TOP2a, and pl6INK4a; and iii) markers of chromosomal instability, such as a gain of chromosome arm 3q and HPV DNA integration [2].
• pl6INK4a GenBank Accession number L27211.
• pl6INK4a overexpression is a result of inactivation of the cell-cycle regulatory retinoblastoma protein (pRb) by High Risk HPV E7. [3-6].
• pl6INK4a detection has now multiple clinical indications, in the screening and prevention programs, either at cytology or histopathology level [2].
• HPV diagnosis is still based on citology, since the virus clearly alters the infected cells. Precisely, this phenomenon is called koilocytosis and indicates the presence of an HPV infection.
• a koilocyte is a squamous epithelial cell that has undergone a number of structural changes, which occur as a result of infection of the cell by human papillomavirus (REF : Nucci MR, Oliva E., ed. (2009). Gynecologic pathology: A volume in the series - Foundations in diagnostic Pathology. Elsevier Churchill Livingstone). Such morphological changes are not necessarily of displastic, as those present in the Cervical Intraepithelial Neoplasia (CIN).
• CIN Cervical Intraepithelial Neoplasia
• pl6INK4a (pi 6) has been recently proposed as a marker for Cervical Intraepithelial Neoplasia 2 (CIN2), CIN3 or even cancer development within 3 years in HPV-positive women.
• CIN2 Cervical Intraepithelial Neoplasia 2
• CIN3 Cervical Intraepithelial Neoplasia 2
• HPV positive pi 6 positive women would need immediate colposcopy (clinical intervention) to be safe (Carozzi et al. Lancet Oncology 2013, 14(2): 168-76)).
• the pl6 marker is not always effective in detecting CIN2.
• Sumoylation is a post-translational modification where the Small Ubiquitin-like Modifier (SUMO) proteins are reversibly attached to the protein target through an ubiquitin-like pathway, with many different outcomes on protein stability, interaction and localization, DNA repair and replication, transcriptional regulation, cell cycle control, apoptosis, cell signalling and viral replication [7, 8].
• viruses are also known to target post- translational modification systems, such as the SUMO pathway [9].
• most of the DNA viruses parvoviruses, adenoviruses, papoviruses and herpesviruses
• DNA viruses parvoviruses, adenoviruses, papoviruses and herpesviruses
• viral proteins that are sumoylated or interact with SUMO components.
• Conjugation of target proteins with SUMO requires a series of events catalyzed by the El (SUMO- activating SAE1/SAE2) (e.g., GenBank Accession numbers (SAE1): NM_001145714, SUMO 1 -activating enzyme subunit 1 transcript variant 3; NR_027280, SUMO 1 -activating enzyme subunit 1 transcript variant 4; NM_005500, SUMO 1 -activating enzyme subunit 1 transcript variant 1; NM_001145713, SUMOl- activating enzyme subunit 1 transcript variant 2; Homo sapiens ubiquitin-like modifier activating enzyme 2 (UBA2 or SAE2), NM_005499), E2 (SUMO- conjugating Ubc9) (e.g., GenBank Accession numbers: NM 003345, Homo sapiens ubiquitin-conjugating enzyme E2I (UBE2I), transcript variant 1; NM l 94259, Homo sapiens ubiquitin
• SUMOl Human tissues express four SUMO family members, SUMOl (including GenBank Accession numbers NM 001005782, NM_003352, NM_001005781, NR_002189, NR_002190), the nearly identical SUM02 (GenBank Accession numbers NM 001005849, NM 006937) and SUM03 (GenBank Accession number NM 006936), all ubiquitously expressed, and SUM04 (GenBank Accession number NG 012301), which is highly similar to SUM02 but primarily expressed in kidney, lymph node and spleen [7, 8]. The mechanisms regulating paralog-selective sumoylation are still not well defined, although SUMO E3 enzymes are the likely candidates to regulate the preferential usage of SUMOl over SUM02/3 in the cells.
• Heaton PR. et al. (Virus Res. 201 1 Jun; 158(1-2): 199-208) demonstrated that high risk HPV E6 oncoproteins are capable of targeting the sumoylation system through reducing the SUMO conjugating enzyme, Ubc9, levels. The authors thus asked whether HPV could affect components of the SUMO pathway. Indeed, the present studies in cervical biopsies and in an in vitro experimental models show that HPV16 E6 and E7, contrary to what is shown in Heaton et al., 2011, have the ability to induce the up-regulation of enzymes involved in the sumoylation machinery, in particular Ubc9. The oncoproteins also diversely affect the two main SUMO paralogs, namely SUMOl and SUM02/3.
• SENP1 SUMO-specific protease 1, member of de-SUMOylation protease family expression
• Modulating SENP1 level dictates colony formation of prostate cancer cell lines, tumor growth in nude mice and also prostate cancer cell migration and invasion.
• Silencing SENP1 level in highly metastatic prostate cancer cells perturbs their ability to metastasize to the bone and initiates secondary tumors.
• MMP2 matrix metalloproteinase 2
• MMP9 matrix metalloproteinase 2
• WO2010051261 refers to compositions and methods for the differential detection of high risk forms of HPV from a urine sample provided by a patient. Specifically, the invention refers to primers and probes that specifically recognize and bind sequences within the El gene of HPV. Detection of high risk forms of HPV identify individuals at risk of developing or in the early stages of cervical carcinoma.
• KR 20090102318 relates to a kit for diagnosing breast cancer using UBC9 gene or Ubc9 protein provided to measure the expression amount of Ubc9 protein and diagnose the breast cancer.
• WO2011/130660 supports the use of SUMO as a marker of cancer development and target for cancer therapy, in particular for sarcoma, carcinoma, lymphoma, glioblastoma, or astrocytoma, brain cancer. No data on cervical cancer are provided.
• WO 03/025543 refers to methods of diagnosing whether an epithelial tissue is an abnormal tissue by determining an expression pattern for Promyelocytic leukemia protein (PML) in the epithelial tissue; determining an expression pattern for nuclear bodies in the epithelial tissue; determining SUMO-1 co-localization and comparing the expression pattern for PML and the expression pattern for nuclear bodies with a control.
• PML Promyelocytic leukemia protein
• the authors thus envisage the use of proteins involved in the SUMO pathway, as e.g. the conjugating enzyme Ubc9, SUMOl, SUM02/3, SAE-1, SAE-2, SUMO E3 ligases, SUM04 and PIAS protein family, as a biomarker of HPV oncogenic infection.
• the present immunohistochemistry (IHC) data show that the detection of at least one protein belonging to the SUMO pathway, e.g.
• Said biomarkers can be used either alone or in combination with other markers of HPV oncogenic infection, e.g. pl6 (pl6INK4a).
• pl6 pl6INK4a
• Authors herein show that Ubc9 staining can spot CIN2 cases missed by pl6INK4a staining (Tables 6-7). The same applies also to CINl and CIN3.
• An object of the present invention is a biomarker belonging to the group of at least one:
• the HPV infection is an high-risk HPV infection, more preferably causative of cervical cancer.
• HPV infection is a low-risk HPV infection, more preferably causative of condyloma.
• the biomarker as above defined belong to the group of: Ubc9, SUMOl, SUM02/3, SAE-1, SAE-2, SUMO E3 ligases, SUM04 and/or PIAS protein family.
• the biomarker is Ubc9, SUMO land/or SUM02/3. Even more preferably the biomarker is Ubc9.
• Another object of the invention is an in vitro method for assessing the risk and/or for diagnosing and/or for prognosing and/or for monitoring the progression of an HPV infection comprising the steps of:
• the in vitro method for monitoring the progression of an high- risk HPV infection comprises the steps of:
• step a) measuring the alteration of the amount of said biomarker in said test sample and b) comparing the measured amount of step a) with an appropriate control amount.
• Said alteration can be an increase or a decrease in the amount of the biomarker.
• An increase can be correlated to an aggravation of the infection, which in case of high- risk HPV infections can lead to a cervical cancer, or to an aggravation of the cervical cancer.
• a decrease can be correlated to an amelioration of the infection or to recovery of the subject.
• step a) If by comparing the measured alteration of the amount of step a) with the appropriate control amount of said biomarker, the alteration of the amount of said biomarker in the test sample corresponds to an increase, the subject of step a) may go towards an aggravation of the infection, which can lead to a cervical cancer, or to an aggravation of the cervical cancer, in case of high-risk HPV infections.
• step a) If by comparing the measured alteration of the amount of step a) with the appropriate control amount of said biomarker, the alteration of the amount of said biomarker in the test sample corresponds to a decrease, the subject of step a) may go toward an amelioration of the infection or to recovery.
• the in vitro method for assessing the risk and/or diagnosing and/or prognosing an HPV infection comprises the steps of:
• step b) comparing the measured amount of step a) with an appropriate control amount, wherein an amount of said biomarker in the test sample higher than the control amount indicates that the subject is either at increased risk for developing or is affected by an HPV infection.
• the HPV infection is an high-risk HPV infection, possibly causative of cervical cancer.
• the HPV infection is a low-risk HPV infection, possibly causative of condyloma.
• the detection of the biomarker is preferably correlated with the presence of HPV-related lesions, which are preferably condyloma, Low grade Squamous Intraepithelial Lesions (LSILs) or High grade Squamous Intraepithelial Lesions (HSILs).
• HPV-related lesions which are preferably condyloma, Low grade Squamous Intraepithelial Lesions (LSILs) or High grade Squamous Intraepithelial Lesions (HSILs).
• An amount of said biomarker in the test sample higher than the control amount can indicate that the subject of step a) presents HPV-related lesions and is either at increased risk for developing or is affected by an HPV oncogenic infection and/or cervical cancer.
• the amount of other different biomarkers of HPV oncogenic infection can also be measured and compared with an appropriate control amount of the same biomarkers.
• the in vitro method according to the invention preferably further comprises the measure of the amount of pl6 (pl6INK4a) and the comparison with an appropriate control amount of pi 6.
• pl6 (pl6INK4a) amount can be the amount of the respective protein or mR A.
• a further object of the invention is a kit for assessing the risk and/or diagnosing and/or prognosing and/or monitoring the progression of an HPV infection, comprising:
• Control means can be used to compare the increase of amount of the biomarker to an appropriate control value.
• the control value may be obtained for example, with reference to known standard, either from a normal subject or from normal population.
• the means to detect and/or measure the amount and/or measure the alteration of the amount of at least one biomarker as above defined are preferably at least one antibody, functional analogous or derivatives thereof.
• Said antibody, functional analogous or derivatives thereof can be specific for said biomarker.
• the detection and/or the measurement of the amount and/or the measurement of the alteration of the amount may be referred to one, two, three, four, five, six, seven, eight, etc biomarkers as above defined. Any combination of said biomarkers is suitable for the purpose of the invention.
• the kit of the invention comprises:
• kits for assessing the risk and/or diagnosing and/or prognosing and/or monitoring the progression of an HPV infection comprising:
• kits according to the invention can further comprise customary auxiliaries, such as buffers, carriers, markers, etc. and/or instructions for use.
• the above biomarker can detect the presence of HPV-related lesions, preferably condyloma, Low grade Squamous Intraepithelial lesions (LSILs) (Cervical Intraepithelial Neoplasia 1 (CIN 1)) and/or High grade Squamous Intraepithelial Lesions (HSILs) (Cervical Intraepithelial Neoplasia 2/3 (CIN 2/3)).
• LSILs Low grade Squamous Intraepithelial lesions
• HSILs High grade Squamous Intraepithelial Lesions
• the "appropriate control amount” may be the amount quantified, measured or assessed in a sample isolated from a healthy subject or from a patient affected by another infection or from a subject not affected by high-risk HPV infection and/or cervical cancer.
• the appropriate control amount may be the amount quantified, measured or assessed in a sample isolated from the same subject at various time point e.g. before the start of a therapy, at various time point throughout the course of the therapeutic agent, etc.
• test sample of the in vitro methods according to the present invention can be a cell and/or tissue sample and/or a biological fluid.
• the in vitro methods according to the present invention can be carried out e.g. in FFPE (Formalin-fixed-paraffin-embedded) samples or in a suspension colorimetric assay.
• FFPE Formmalin-fixed-paraffin-embedded
• step a) is preferably carried out by immunohistochemistry, cytology, ELISA, or spectrofluorometry.
• HPV16 and 18 infections can lead to HPV oncogenic infections.
• the expression "detecting" in relation to a protein or a nucleic acid refer to any use of any method of observing, ascertaining or quantifying signals indicating the presence of the target protein or nucleic acid in a sample or the absolute or relative quantity of that target protein or nucleic acid in a sample.
• Methods can be combined with protein or nucleic acid labeling methods to provide a signal, for example: immunohistochemical staining, ELISA, cell suspension, cytology, fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or adsorption, magnetism, enzymatic activity and the like.
• the expression "measuring the amount” can be intended as measuring the amount or concentration or level of the respective protein and/or mRNA thereof, preferably semi-quantitative or quantitative.
• Measurement of a protein biomarker can be performed directly or indirectly.
• Direct measurement refers to the amount or concentration measure of the biomarker, based on a signal obtained directly from the protein, and which is directly correlated with the number of protein molecules present in the sample.
• This signal - which can also be referred to as intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of the biomarker.
• Indirect measurements include the measurement obtained from a secondary component (e.g., a different component from the gene expression product) and a biological measurement system (e.g.
• amount refers but is not limited to the absolute or relative amount of proteins, and any other value or parameter associated with the same or which may result from these.
• values or parameters comprise intensity values of the signal obtained from either physical or chemical properties of the protein, obtained by direct measurement, for example, intensity values in an immunoassay, mass spectroscopy or a nuclear magnetic resonance. Additionally, these values or parameters include those obtained by indirect measurement, for example, any of the measurement systems described herein.
• Methods of measuring mR A in samples are known in the art.
• the cells in a test sample can be lysed, and the levels of mRNA in the lysates or in RNA purified or semi-purified from lysates can be measured by any variety of methods familiar to those in the art.
• Such methods include hybridization assays using detectably labeled DNA or RNA probes ( i.e., Northern blotting) or quantitative or semi-quantitative RT-PCR methodologies using appropriate oligonucleotide primers.
• RNA protection assay R A
• cDNA and oligonucleotide microarrays R A
• representation difference analysis RDA
• differential display EST sequence analysis
• SAGE serial analysis of gene expression
• variant refers to a protein substantially homologous to the protein involved in the sumoylation pathways as above defined.
• a variant includes additions, deletions or substitutions of amino acids.
• variant also includes protein resulting from post-translational modifications such as, without limitation, glycosylation, phosphorylation or methylation. Examples of variants are disclosed at pp. 2 and 3. Nucleic acid encoding or translating such variants are included in the scope of protection of the invention, as well.
• At least one biomarker as above defined may be detected in the same test sample in the in vitro methods of the invention, said biomarker being selected from the group of: Ubc9, SUMOl, SUM02, SUM03, SAE-1, SAE-2, SUMO E3 ligases, SUM04, PIAS protein family and/or any other protein involved in the sumoylation pathway, preferably Ubc9, SUMOl, SUM02, SUM03, SAE-1 and/or SAE-2, more preferably Ubc9 and/or SUMO 2/3, even more preferably Ubc9.
• Ubc9, SUMOl, SUM02, SUM03, SAE-1 and/or SAE-2 more preferably Ubc9 and/or SUMO 2/3, even more preferably Ubc9.
• Two biomarkers may be detected, said biomarkers being selected from the group of: Ubc9, SUMOl, SUM02, SUM03, SAE-1, SAE-2, SUMO E3 ligases, SUM04, PIAS protein family and/or any other protein involved in the sumoylation pathway. Any combination is suitable for the purpose of the invention, for instance detecting, e.g. Ubc9 and SUM02, Ubc9 and SUMOl, or SUMOl and SUM02, etc.
• biomarkers may be detected in the same test sample, said biomarkers being selected from the group of: Ubc9, SUMOl, SUM02, SUM03, SAE-1, SAE-2, SUMO E3 ligases, SUM04, PIAS protein family and/or any other protein involved in the sumoylation pathway. Any combination is suitable for the purpose of the invention, for instance measuring Ubc9, SUMOl and SUM02, or measuring Ubc9, SUM02 and SUM03, etc...
• biomarkers may be measured in the same test sample, said biomarkers being selected from the group of: Ubc9, SUMOl, SUM02, SUM03, SAE-1, SAE-2, SUMO E3 ligases, SUM04, PIAS protein family or any other protein involved in the sumoylation pathway. Any combination is suitable for the purpose of the invention, for instance measuring Ubc9, SUMOl, SUM02, and SUM03, or measuring SUMOl, SUM02, SUM03, SAE-1, etc.
• biomarkers may be detected in the same test sample. Any combination is suitable for the purpose of the invention.
• biomarkers may be measured in the same sample, said biomarkers being selected from the group of: Ubc9, SUMOl, SUM02, SUM03, SAE-1, SAE-2, SUMO E3 ligases, SUM04, PIAS protein family or any other protein involved in the sumoylation pathway.
• the in vitro methods of the invention can also be used to follow the course of a HPV oncogenic infection and/or cervical cancer, or to follow the effect of a therapeutic treatment on HPV oncogenic infection and/or cervical cancer, thus e.g. monitoring the progression of an HPV oncogenic infection.
• the appropriate control amount is quantified in the same subject before pharmacological treatment or at different time points.
• the in vitro methods and kits of the invention can assess the risk, diagnose or prognose or monitor the progression of cancer caused by HPV, in particular by high- risk HPV, such as HPV16 and 18, as e.g. cervical cancer.
• the present invention also refers to a method for prevention of cancer caused by HPV, preferably by high-risk HPV, as e.g. cervical cancer, comprising the identification or detection of HPV-related lesions, preferably CINl and/or CIN2/3, with at least one biomarker as above defined. Once the lesions have been identified they can be eradicated.
• HPV high-risk HPV
• HPV-related lesions preferably CINl and/or CIN2/3
• biomarker as above defined.
• the biomarker as described in the present invention can be used to prevent cancer caused by HPV, preferably by high-risk HPV, as e.g. cervical cancer.
• FIG. 1 Scoring for Ubc9 and SUM02.
• the graphs show the number of samples, expressed as percentage on total samples analyzed, that scored positive or negative for either Ubc9 or SUM02. Some of them were not assessable (indicated as NA in figure).
• 18 Formalin-Fixed, Paraffin-Embedded (FFPE) samples from Condyloma (panel A), 32 from CINl patients, 35 from CIN2 and CIN3 patients (panel B) were scored for Ubc9 positivity, based upon the intensity of the colouring and the percentage of cells staining positive, giving a score from basal/negative to positive (+) or very positive (++). Basal indicates a basal colouring for the antibody (normal tissue staining).
• FFPE Paraffin-Embedded
• FIG. 1 Analysis of Ubc9 expression in HPV16-transduced Human Keratinocytes (HKs).
• FIG. 1 SUMO-1 in HPV16-transduced primary Human Keratinocytes (HKs).
• FIG. 1 SUMO-2/3 in HPV16-transduced primary Human Keratinocytes (HKs).
• E6E7 HPV16 proteins, and SUMO-2/3 or GAPDH mRNA levels were measured by quantitative RT-PCR.
• FIG. 9 PIASy expression in HPV16-transduced Human Keratinocytes (HKs).
• KSFM Keratinocyte Serum- Free Medium
• human keratinocytes were isolated from skin biopsies and grown in Keratinocyte Serum- Free Medium (KSFM, Gibco) as described previously [15]. Briefly, human keratinocytes were isolated from skin by Dispase lOU/ml (Gibco) digestion for 16 hours at 4°C to remove the epidermis, followed by a trypsinization (Trypsin 500mg/l, Versene, 30 minutes at 37°C) step, used to obtain the isolated cells. Primary cultures were then established in KSFM containing bovine pituitary extract (BPE, 30 micrograms/ml, Gibco) and epidermal growth factor (EGF, 0.2 ng/ml, Gibco). Cells were used from passage 2 to 5 for the retroviral transduction.
• BPE bovine pituitary extract
• EGF epidermal growth factor
• the plasmids pLXSN, pLXSN-HPV16E6, pLXSN-HPV16E7, and pLXSN- HPV16E6E7 were kindly provided by M. Tommasino (IARC, Lyon, France). Retroviral supernatants generated by transfection of the pLXSN constructs into Phoenix ampho cells were used to transduce primary human keratinocytes, as previously described[16]. Twenty- four hours after transduction, keratinocytes were selected with 0.1 micrograms/ml G418 (Gibco) for 1 week.
• RNA from G418-selected keratinocytes was extracted with the RNeasy mini kit (Qiagen), following the manufacturer's instruction. Residual contaminating DNA was removed with the RNase-free DNase set (Qiagen). RNA was reversely transcribed using the ImProm-II system for RT-PCR (Invitrogen). Real-time quantitative RT- qPCR for UBC9, SUMO-1 and SUMO-2/3 was performed with the ABI Prism 7900 Sequence Detection System, using the Fast SYBR Green Master Mix (Applied Biosystems) and primers 5 '-ACAGTGTGCCTGTCCATCTTAG-3 ' (forward) (SEQ ID No. l) and 5 '-TGTTTTGGCAGTAAATCGTGTA-3 ' (reverse) (SEQ ID No.2), 5 '-TGTCAAAGACAGGGTGTTCCAA-3 ' (forward) (SEQ ID No.3) and
• G418-selected keratinocytes were lysed with El A Buffer (50mM Hepes, 250mM NaCl, 0.1% NP-40 and cocktail of protease inhibitors, Sigma Aldrich). Total proteins (30 micrograms) were resolved by 16%> SDS-PAGE under reducing conditions, and electroblotted with antibodies raised against Ubc9 (Abeam, no. 21193, 1 :5000 dilution), SUMO-1 (Sigma Aldrich, S8070, 1 :3000 dilution), SUMO-2/3 (Abeam, AB22654, 1 :5000 dilution), SAE1 (Abeam, no.
• cervical samples were formalin- fixed and paraffin- embedded according to the established procedures. All sections were counterstained with Mayer's haematoxylin and visualized using a bright field microscope. Images were generated with a Leica DFC280 Microscope.
• anti-Ubc9 (Santa Cruz, SC10759) (Antigen retrieval at 99 C for 40 minutes in water bath, EDTA buffer pH 8.0, cool down to R.T. for 20 minutes), 1 : 1000, incubation O/N at 4 C; anti-SUM02/3 (Abeam, AB22654) (Antigen retrieval at 99 C for 40 minutes in water bath, Sodium Citrate buffer pH 6.0, cool down to R.T. for 20 minutes), 1 : 1000, incubation O/N at 4 C.
• the Envision kit from DAKO was utilized and after the chromogenic visualization step using the 3,3'- Diaminobenzidine (DAB) chromogen, slides were counterstained with hematoxylin and coverslipped.
• DAB 3,3'- Diaminobenzidine
• the CINtec pl6-INK4A Cytology kit (Dako Cytomation, now Roche mtm laboratories AG, Heidelberg, Germany) was used, which applies a pl6-specific monoclonal antibody (clone E6H4).
• anti-Ubc9 (Santa Cruz, SC10759) (Antigen retrieval at 99 C for 15 minutes in water bath, EDTA buffer pH 8.0, cool down to R.T. for 20 minutes), 1 :3200, incubation O/N at 4 C.
• the DAKO kit (LSAB+ System-HRP) was utilized and after the chromogenic visualization step, slides were counterstained with hematoxylin and coverslipped. Concordance Analysis
• Three independent pathologists reviewed the slides blinded at the original diagnosis; the diagnostic concordance was analyzed overall on all diagnostic categories (negative; CINl, including condyloma; CIN2; CIN3), or grouping the diagnosis into three categories (Neg: negative; CINl, including condyloma; CIN2+: CIN2 and CIN3) or into two categories (CIN2-: less than CIN2; CIN2+: CIN2 or CIN3). The results were plotted into tables (Tab. 1-5). The concordance is expressed as percentage of exact agreement.
• CIN cervical intraepithelial neoplasia
• HSILs High grade Squamous Intrapethelial Lesions
• LSILs Low grade Squamous Intraepithelial Lesions
• CINl refers to LSILs
• CIN2 and 3 to HSILs.
• the authors analyzed low grade SILs, high grade SILs, and condyloma. All high grade lesions showed a highly statistically significant increase of SUM02 proteins and Ubc9 enzymes as compared to normal tissue ( Figure 1 and Figures 3 and 4).
• HPV16 E6 and E7 oncoproteins affect SUMO pathway components.
• Ubc9 is different from pl6INK4a.
• pl6INK4a is an established HPV surrogate biomarker.
• pl6 test CINtec pl6 kits from MTM/ROCHE
• pl6INK4a overexpression is a result of inactivation of the cell-cycle regulatory retinoblastoma protein (pRb) by High Risk HPV E7.
• Tables 1-2 Concordance with histological diagnosis (hematoxylin and eosin) of three different pathologists (Ope 1-3) after Ubc9 (Table 1) and pl6 (Table 2) staining. Results are expressed for all diagnosis categories; subgrouped into three categories, namely negative, CINl, CIN2+ (CIN2 or CIN3); and subgrouped into two categories CIN2- (less than CIN2) and CIN2+.
• the data show an increase in concordance after Ubc9 staining, more evident than after pl6 staining.
• Table 3 Data show the concordance of Ubc9 scorings. Results are expressed for all diagnosis categories; subgrouped into three categories, namely negative, CINl, CIN2+; and subgrouped into two categories CIN2- and CIN2+. The data show better concordance grouping in CIN2- and CIN2+.
• Table 5 Concordance of Ubc9 vs pl6 stainings. The data show that the two markers are different.
• Tables 6-7 Data are based on Ubc9 vs pl6 reading on 65 patients.
• Ubc9 is a different marker, when compared to pl6INK4a which is only upregulated by E7 oncoprotein expression, making it a novel diagnostic tool.
• LSILs and HSILs are the most important and determinant key to cervical cancer prevention.
• Today's strategy implies both the identification and the eradication of CIN2/3 to prevent invasive cancer and to monitor LSILs towards clearance or possibile progression to HSILs.
• the trigger to treatment is the histopatho logical diagnosis of CIN2/3, which the present data are indicating as very sensitive to proteins involved in the sumoylation pathway, in particular Ubc9 staining.
• Ubc9 can also detect CINl, thus making it a valuable marker to avoid overdiagnosis and therefore over-treatments.
• Hay RT. SUMO a history of modification. Mol Cell 2005; 18(1): 1-12.
• Livak KJ, Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-8.

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