METHOD FOR DETECTING CARCINOGENESIS IN THE UTERINE CERVIX

Abstract

The invention concerns some methods for diagnosing cancer of the uterine cervix (or extra-uterine) which integrate or surrogate the field of action of cytology with that of molecular biology, and provide a system for diagnosis, prognosis and management of HPV-induced cervical lesions (or extra-uterine) which exploit, in particular, the analytical methods of Western Blot and Sandwich ELISA in order to detect those cases where the transformation towards a neoplastic lesion has become irreversible. The methods consist in detecting, in samples of cells taken from the squamous-columnar junction of the uterine cervix of a patient under examination, the proteins encoded by viral oncogenes E6 and E7 and those encoded by the tumor suppressor genes of the host cell p53 and pRB, and in detecting by Western Blot and/or by Sandwich ELISA the possible interaction between proteins E6 and p53, and between proteins E7 and pRb, as an index of irreversible transformation towards a neoplastic lesion.

Claims

1. A method for detecting a neoplastic cell transformation induced by human Papillomavirus (HPV) in women undergoing a screening for uterine cervix carcinoma, comprising detecting, in samples of cells taken from the squamous-columnar junction of the cervix epithelium of a patient being examined, the presence of a protein complex E6/p53 made by the protein E6 with the protein p53 and/or a protein complex E7/pRB made by the protein E7 with the protein pRB, wherein the detection of said two protein complexes shows that in the patient under examination the carcinogenesis has become irreversible, said detection being performed by the combined use of antibodies anti-protein E6 and antibodies anti-protein E7.

2. A method according to claim 1, comprising the following steps according to the Western Blot technique: a) preparing a sample of cells taken from the squamous-columnar junction of the cervix epithelium of a patient being examined, and placing it in physiological solution; b) extracting proteins from said sample and denaturizing the proteins obtained; c) subjecting the so obtained denatured proteins to electrophoresis on polyacryl amide gel; d) transfening the proteins obtained from the previous step on a membrane and neutralizing the free sites of said membrane; e) identifying said proteins by incubating them with at least the following antibodies: primary monoclonal antibody anti-protein E6; primary monoclonal antibody anti-protein E7; and then incubating with secondary anti-lgG antibodies conjugated with biotin or with a messenger enzyme such as alkaline phosphatase; f) subjecting the products from the previous step to colorimetric detection or to detection by chemiluminescence; wherein the results of said detection which show a double reaction band for the proteins E6 and E7, said double band comprising a first band corresponding to the molecular weight of the protein sought, i.e. E6 or E7, and a second band corresponding to a molecular weight distinct and higher than the molecular weight of the respective protein of the first band, said molecular weight being substantially equal to 71 kD for the protein complex E6/p53 and to 115.5 kD for the protein complex E7/pRB, indicate that in the patient examined carcinogenesis has become irreversible.

3. A method according claim 2, wherein in said step e) said proteins are also incubated with the following anti-bodies: primary monoclonal antibody anti-protein p53; primary monoclonal antibody anti-protein pRb; the further steps being similar to those carried out for the case of incubation with said primary monoclonal antibodies anti-protein E6 and anti-protein E7, and Wherein the results of said detection showing a double reaction band for at least one of the proteins p53 and pRb, said double band comprising a first band corresponding to the molecular weight of the protein sought, p53 or pRb, and a second band corresponding to a molecular weight distinct and higher than the molecular weight of the respective protein of the first band, indicate that in the patient examined carcinogenesis has become irreversible.

4. A method according to claim 2, wherein the results of said detection showing a single reaction band for both proteins 136 and E7, said single band corresponding to the molecular weight of the protein sought E6 or E7, indicate that in the patient examined carcinogenesis is not yet started and has not become irreversible.

5. The method according to claim 4, wherein the results of said detection which do not show any reaction band for proteins E6 and E7 indicate that in the patient examined no integration of viral DNA with cellular DNA has occurred.

6. A method according to claim 2, wherein the secondary anti-IgG antibodies of said operation e) are conjugated with alkaline phosphatase, and wherein said detection of operation f) is a colorimetric detection or a detection by chemiluminescence.

7. A method according to claim 2, wherein said primary monoclonal antibody anti-protein E6 is a mouse anti-protein E6 antibody of the viral types HPV16 and HPV18, and said primary monoclonal antibody anti-protein E7 is a mouse anti-protein E7 antibody of the viral types HPV16 and HP V18.

8. The method according to claim 3, wherein said primary monoclonal antibody anti-protein p53 is a mouse monoclonal anti-human protein p53 antibody, and said primary monoclonal antibody anti-protein pRb is a mouse monoclonal anti-human protein pith antibody.

9. A method according to claim 1, wherein said detection is performed by the combined use of antibodies anti-protein E6, antibodies anti-protein E7, antibodies anti-protein p53 and antibodies anti-protein pRB, comprising the following steps according to the Sandwich ELISA technique: a) identifying a sample to be examined, stored according to liquid phase cytology (LBC); b) adsorbing, in different wells of a microplate, antibodies anti-protein E6 and anti-protein E7, as capture anti-bodies of viral protein E6 or E7; c) blocking the remaining sites of the well bonding the proteins by a blocking buffer; d) dispensing directly into each well the indicated cell sample to be analyzed; e) identifying the proteins of interest by incubation with diluted antibody anti-protein p53 in the wells adsorbed with antibody anti-protein E6 and by diluted antibody anti-protein pRB in the wells adsorbed with antibody anti-protein E7; f) blocking the endogenous substrate; g) incubating into the respective wells the antibody conjugated to horseradish peroxidase (HRP); h) dispensing a chromogen solution in each well i) subjecting the step h) reaction product to reading of the light intensity detected at 450 nm wavelength, by spectrophotometer, wherein results showing the occurred staining, documented by a positive absorbance, indicate that in the patient under examination protein complex E6/p53 and/or E7/pRB are present, confirming interaction between viral and human proteins and therefore indicating that in that patient under examination the carcinogenesis started and has become irreversible.

10. (canceled)

11. A method according to claim 9, wherein in case the detection results do not show any reaction positivity, with absence of signal absorbance at 450 nm, for both protein complexes E6/p53 and E7/pRB, the result indicates that in the patient under examination no integration of viral DNA with cellular DNA has occurred; not excluding the possibility of being in presence of integration of viral DNA with the cell DNA.

12. A method according to claim 9, wherein said primary monoclonal antibody anti-protein E6 is a mouse monoclonal antibody anti-protein E6 of the viral types HPV16 and HPV18, said primary monoclonal antibody anti-protein P7 is a mouse monoclonal antibody anti-protein E7 of the viral type HPV16, said primary monoclonal antibody anti-protein p53 is a mouse monoclonal antibody anti-human p53 protein, said primary monoclonal antibody anti-pRB protein is a mouse monoclonal antibody anti-human pRB protein.

13. A method according to claim 2, wherein said method is inserted in the frame of a screening program of a female population, or in a program for early diagnosis, for the detection of uterine cervix carcinoma, after a cytological diagnosis of atypical squamous cells (ASC) or worse, and a subsequent positive result of the hr-HPV DNA test.

14. A method according to claim 2, wherein said method is inserted in the frame of a screening program of a female population, or in a program for early diagnosis, for the detection of uterine cervix carcinoma, after a positive result of the hr-HPV DNA test, and subsequent cytological diagnosis of atypical squamous cells (ASC) or worse.

15. A method according to claim 2, wherein said method is inserted in the frame of diagnostic activity, screening or follow-up of patients with neoplastic lesions HPV induced, or suspected.

16. A method according to claim 1, wherein said detection is performed by the combined use of antibodies anti-protein E6, antibodies anti-protein E7, antibodies anti-protein p53 and antibodies anti-protein pRB, comprising the Wowing steps according to the Sandwich ELISA technique: a) identifying the proteins of interest by incubation with diluted antibody anti-protein p53 in the wells adsorbed with antibody anti-protein E6 and by diluted. antibody anti-protein pRB in the wells adsorbed with antibody anti-protein E7; b) blocking the endogenous substrate; c) incubating into the respective wells the antibody conjugated to horseradish peroxidase (HRP); d) dispensing a chromogen solution in each well wherein commercially available systems are used that allow to amplify the chromogenic signal, preferably using antibodies anti-protein p53 or pRB biotinylated and evidenced with streptavidin conjugated to peroxidase or to another enzyme.

17. A method according to claim 9, wherein said method is inserted in the frame of a screening program of a female population, or in a program for early diagnosis, for the detection of uterine cervix carcinoma, after a cytological diagnosis of atypical squamous cells (ASC) or worse, and a subsequent positive result of the hr-HPV DNA test.

18. A method according to claim 9, wherein said method is inserted in the frame of a screening program of a female population, or in a program for early diagnosis, for the detection of uterine cervix carcinoma, after a positive result of the hr-HPV DNA test, and subsequent cytological diagnosis of atypical squamous cells (ASC) or worse.

19. A method according to claim 9, wherein said method is inserted in the frame of diagnostic activity, screening or follow-up of patients with neoplastic lesions HPV induced, or suspected.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The specific features of the invention, as well as the advantages thereof, will become more apparent referring to the description of the experimental work reported in the following with merely illustrative purposes and to the related figures, wherein:

[0084] FIG. 1 shows a histogram with the cytological diagnosis of 78 cases selected as positive from a population of 2500 women undergoing a screening with Pap-test.

[0085] FIG. 2 shows a histogram with the results of the application of the diagnostic method according to the present invention to the 78 cases selected as positive to the Pap-test of FIG. 1;

[0086] FIGS. 3-6 show the results of Western Blot of one case selected from the results of the diagnostic method according to the invention wherein E6 and E7 protein are present, and wherein E6/p53 and E7/pRb interactions are also present;

[0087] FIG. 7 is a flow diagram schematically illustrating the indications proposed for women management with positive Pap test and positive hr-HPV DNA test for the application of the diagnostic method of the invention; and

[0088] FIG. 8 is a flow diagram schematically illustrating the application of the Sandwich ELISA method to the diagnostic method of the invention.

[0089] Screening Test on a Population of Women Not Selected Undergoing Pap Test as Primary Examination

[0090] In the context of the present invention, a study was carried out aiming at identifying women with clinically relevant lesions that had developed high grade dysplastic lesions or cervical cancer. The study was conducted on 2,500 cervical specimens of women, not selected, which occurred spontaneously at health advisory outpatient clinics centers of Caserta Local Health Board ASL. The research was carried out in the period from June to September 2012 and included information to women and the acquisition of informed consent. In this population conventional Pap tests have been performed and the tests have been analyzed at the Cytopathology Laboratory of Caserta ASL.

[0091] Of the 2,500 women surveyed, a total of 78, that is, 3.12%, were diagnosed not negative, that is, ASC+(Squamous Cells Atypia or worse) to the Pap test. As also shown in the diagram of attached FIG. 1, the cytological diagnoses ASC+were divided as follows: 32 cases were with ASC-US cytology (41%), 42 cases were LSIL (54%) and 4 cases were HSIL (5%).

[0092] 96.88% of cases were negative for squamous intraepithelial lesions or malignant ending in the categories of normal or benign inflammatory changes, sometimes marked by the presence of microorganisms (bacteria, fungi, or trichomonas vaginalis).

[0093] 78 women with positive diagnosis were recalled to undergo a further examination according the method of the present invention, by a second sample to study the integration/interaction of E6/p53 and E7/pRb proteins.

[0094] Sample have been withdrawn and tested according to the procedure described above for the specific embodiment of the method according to the invention, at the Molecular Biology Laboratory of the Marcianise Hospital.

[0095] Data obtained by Western Blot analysis of the 78 samples positive Pap test are shown in the herein attached FIG. 2. The histogram firstly shows that of the 32 samples with ASC-US cytological diagnosis only 2 (6.3%) were positive for presence of while in the other 30 (93.7%) E6 and E7 proteins were not found.

[0096] On the other hand, in all samples with a cytological diagnosis of LSIL (42) and HSIL (4) has been proved the presence of E6 and E7 proteins. In total, therefore, of the 78 cases which have had a not negative diagnosis with Pap tests, only 48 were positive for the presence of the E6 and E7 proteins according to the diagnostic method according to the invention.

[0097] From the data observation, it is clear that in most of ASC-US (93.7%) integration of viral genome did not occur, and as result it may mean: [0098] 1) HPV absent: cytological abnormalities found are only inflammatory in nature; [0099] 2) low-risk HPV present: normally is not able to integrate into genome but is limited to the episomal form;; [0100] 3) high-risk HPV present: it is still in the early stage with very recent infection and lack of integration.

[0101] For all cases with LSIL and HSIL cytological diagnosis were identified E6 and E7 proteins and therefore it can be stated that these samples have a high-risk HPV and certainly the infection is not recent, as already had the viral genome integration.

[0102] Taking in consideration the 48 positive cases with the presence of proteins E6 and E7, also as shown in FIG. 2, the diagnostic method of the present invention allows to observe that the interactions E6/p53 and E7/pRb are present in 4,8% of the LSIL cases and in 75,0% of the HSIL cases, being absent in the ASC-US showing the presence of proteins E6 and E7.

[0103] The results shown in the histogram of FIG. 2 are also summarized in the following table.

TABLE-US-00001 TABLE 1 Correlation of cytological and histological diagnosis of the cases tested with Western Blot for integration/interaction of proteins E6/p53 and E7/pRb. Cytol- Presence No E6/p53 & ogical of E7/pRb Presence of E6/p53 & diag- E6 & E7 interaction E7/pRb interaction nosis proteins Histology Histology ASC- 2 2 2 CIN1-2 0 US (100.0%) LSIL 42 40 37 CIN1 2 1 CIN2 (95.2%) 3 CIN2 (4.8%) 1 CIN3 HSIL 4 1 1 CIN3 3 2 CIN3 (25.0%) (75.0%) 1 CA. SQ. INV. TOTAL 48 43 37 CIN1 5 1 CA. SQ. INV. (89.6%) 2 CIN1-2 (10.4%) 3 CIN3 3 CIN2 1 CIN2 1 CIN3

[0104] The data shown above demonstrate that 48.8% of LSIL and 75.0% of HSIL have the interaction of E6/p53 and E7/pRb leaving to assume that carcinogenesis process is at advanced stage; for remaining LSIL and HSIL cases without interaction, it can be assumed that the carcinogenesis process is still in early stages and still regression is possible.

[0105] The most significant examples of the type of results obtained by Western Blot in the study herein reported are shown in the attached FIGS. 3, 4, 5 and 6, which show the results of Western Blot analysis obtained from samples where in addition to E6 and E7 was also found interaction with p53 and pRb.

[0106] The observation of FIGS. 3 and 4, respectively relating to the Western Blot results with primary monoclonal antibody anti-E6 and anti-E7 protein shows a double stripes of reaction, for each sample, 18 Kd and 71 Kd for anti-E6 protein, 16 Kd and 115.5 Kd for anti-E7 protein 7. The existence of additional stripes of molecular weight much higher those E6 and E7 demonstrates, according to the present invention, the presence of an interaction between E6 and p53 and between E7 and pRb.

[0107] Said hypothesis has been confirmed by two additional Western Blot analysis with primary monoclonal antibodies, respectively, anti-p53 and anti-pRb protein. In FIGS. 5 and 6 are shown the results obtained which confirm such hypothesis, as the reactive stripes exactly match the two additional stripes previously observed.

[0108] For samples with the E6 and E7 proteins but without interaction E6/p53 and E7/pRb, the Western Blot with primary monoclonal antibody anti-E6 protein and anti-E7 protein have shown, for each sample, a single reactive stripe of respectively 18 Kd for the antibody anti-E6 protein and 16 Kd for anti-E7 protein

[0109] For the samples, however, which were negative for the presence of E6 and E7 proteins, both the Western Blot with primary monoclonal antibody anti-E6 protein that with anti-E7 protein showed no reaction stripe, evidence that in these samples there has been no integration of viral DNA into the cell.

[0110] According to the results obtained, it is clear that all HSIL and majority of LSIL studied present the integration of the viral genome with E6 and E7 proteins production. However, 75.0% of high-grade lesions and only few low-grade lesions (4.8%) showed the interaction of proteins E6/p53 and E7/pRb. These data confirm that the interaction is a marker of carcinogenesis.

[0111] Therefore, the Western Blot technique for assessing the presence of E6 and E7 proteins and their possible interaction (E6/p53 and E7/pRb) demonstrates a higher specificity of both HPV DNA test and cytology, allowing identifying patients with clinically relevant lesions (CIN2+). Histological follow-up of LSIL and HSIL have confirmed this hypothesis. In particular (see, Table 1), the four HSIL who had E6/p53 and E7/pRb proteins interaction, two were CIN 3 (Severe DysplasiaCarcinoma in situ) (50.0%) and one invasive squamous cell Carcinoma (25.0%), the fourth case without interaction was diagnosed CIN 3. Also the two LSIL (4.8%) with E6/p53 and E7/pRb proteins interaction are findings one CIN2 (Moderate Dysplasia) (50.0%) and the other CIN 3 (50.0%). The 40 LSIL (95.2%) without interaction were 37 CIN 1 (Mild dysplasia) (88.1%) and 3 CIN2 (7.1%).

[0112] These information confirm that there is a relationship between protein interactions and carcinogenesis, even it is still to understand what are biological bases of step from integrated with those interactive and thus to carcinomatous transformation. Basically only few integrated lesions expressing E6 and E7 proteins overexpression progressing toward complete carcinogenesis. This suggests the hypothesis that there is a quantitative threshold of viral proteins over which their interaction develops. Further studies will be able to confirm this hypothesis according to the most representative case studies.

[0113] Considering the evaluation above reported as fundamental, according to the method of the invention, we propose Western Blot or Sandwich ELISA for carrying out the integration/interaction test of proteins E6/p53 and E7/pRb in diagnostic pathway after hr-HPV DNA positive test and Pap test positive for ASC+ lesions, in particular according the schematic indications in the diagram of FIG. 7.

[0114] Using this diagnostic strategy it is possible increase the specificity of cytology (ASC+) and molecular (hr-HPV DNA positive test) diagnosis in cervical screening, reserving further recall women to the second level of investigation just the cases with integration/interaction of viral genome. The same strategy may be applied also for neoplastic lesions HPV-induced in extra-uterine areas (anus-genital region, oropharyngeal region, etc.).

[0115] An efficient vaccine strategy combined with organized screening and adequate sensitivity and increasing specificity of used tests, as the one here reported as an example, will lead to the decrease of papillomavirus infection prevalence by resulting in decreased impact of cervical cancer, health and socio-economic advantage for society, greatly reducing mortality, also by targeted therapy.

[0116] A further possible advantageous use of the method according to the invention consists in the use of the same method for making experiments, in the various phases of the disease progression, about the activities of anticancer drugs and the inhibiting activity of the interaction E6/p53 and E7/pRb as nicandrenone (Shaikh F. et al. 2012, loc. cit.), for the develop and experiment of said drug in targeted therapy.

[0117] The same methods are to be intended also to identify, in cells samples taken from any anatomical site other than the uterine cervix of any patient under examination, the proteins encoded by oncogenic genes, also non-viral, and those encoded by tumor suppressor genes in the cell examination and to determine, by Western Blot and/or ELISA Sandwich, the possible interaction between them.

[0118] The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.