Ignite Creation Date:
2024-05-06 @ 8:19 PM
Last Modification Date:
2024-10-26 @ 3:25 PM
Study NCT ID:
NCT06339684
Status:
ACTIVE_NOT_RECRUITING
Last Update Posted:
2024-04-03
First Post:
2024-03-25
Brief Title:
HPV Immunological Markers of Cervical Persistent Infection and Oncogenesis
Sponsor:
Fondazione IRCCS Policlinico San Matteo di Pavia
Organization:
Fondazione IRCCS Policlinico San Matteo di Pavia
Study Overview
Official Title:
HPV Immunological Markers of Cervical Persistent Infection and Oncogenesis
Status:
ACTIVE_NOT_RECRUITING
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
HPVImmuno
Brief Summary:
The aim of this observational study is to build an immunological assay to quantify an immunoscore system for clinical practice which could identify HPV lesions with a risk of persistent cervical infection which represents the main predictive factor of neoplastic evolution A pattern of host immunological factors and HPV-related parameters in order to identify an algorithm of risk stratification and tailoring treatment will be identified Finally in patients with HPV infection a virus specific immunity after vaccination will be quantified in order to highlight those patients who have the most significant risk of infection persistence
Detailed Description:
The end point of this study is to analyze host factors including virus-specific immunity and HPV-related parameters influencing the onset and progression of squamous intraepithelial lesion SIL in order to define risk stratification and tailoring treatment developing widely accessible and non-invasive assays immunoscore with high predictive value
The progression of squamous intraepithelial lesion H SIL at 2 years in women with abnormal pap smear will also be evaluated primary end point and the immune host profile after anti-HPV prophylactic and therapeutic vaccination in women with squamous intraepithelial lesions and with persistence of infection will be analyzed secondary end point
The investigators will analyze the patients enrolled prospectively with abnormal pap smear every six months up to two years after diagnosis At each time point during colposcopy peripheral blood 30 mL and cervical samples vaginal washing cervical biopsy and pap-smear will be collected for the characterization of local and peripheral immune response Viral load HPV integration will be assessed on cytological and tissue samples obtained from the patients at time of diagnosis andor curative surgery
For the secondary endpoints the investigators will enroll patients with confirmed HPV infection undergoing therapeutic and prophylactic HPV vaccination The vaccination will be performed immediately after HPV lesion diagnosis Follow-up program implies colposcopy every six months for 2 years The investigators will analyze HPV-specific immune response every six months and viral load and HPV integration at the time of diagnosis and at the end of the follow-up
To evaluate antigen-specific proliferative response PBMCs will be stimulated in triplicate in 96-well round-bottom plates with antigens L1 E6 E7 proteins of HPV- 16 and -18 and human Actin peptide pools for 7 days Culture medium was RPMI 1640 supplemented with 2 mM L-glutamine 100 UmL penicillin and 100 µgmL streptomycin solution 10 of heat inactivated FBS 1 mM Sodium Pyruvate 100 µM non-essential amino acids and 50 µM 2-Mercaptoethanol After culture cells will be washed stained with LiveDead Fixable Violet Dye and subsequently with CD3 CD4 CD8 CD25 CD278 ICOS Finally cells will be washed and resuspended in PBS 1 paraformaldehyde
A Cell Proliferation Index CPI for antigen-specific expanded T-cells will be determined by subtracting the percentage of CD25ICOS CD3CD4 or CD3CD8 detected in PBMC incubated with actin peptides from the percentage of CD25ICOS T-cell subsets detected in PBMC incubated with HPV peptides A CPI 15 will be considered negative while a value 15 will be considered positive
Flow cytometry analyses were performed with a FACS Canto II flow cytometer and BD DIVA software
For the analysis of HPV-related features molecular approaches will be used HPV DNA will be extracted from biopsies and lesion swabs using automatized systems after specific lysis treatment for tissues
HPV DNA will be then quantified using real time PCR specific for L1E2 and E6 regions in detail MY 0911 fragment of L1 gene will be used Results will be expressed as the number of copies100000 cells and normalized using a housekeeping gene
For the differential quantification of integrated or episomal HPV DNA a specific standardized system for the amplification of ORF E2 and E6 will be used
The progression of squamous intraepithelial lesion H SIL at 2 years in women with abnormal pap smear will also be evaluated primary end point and the immune host profile after anti-HPV prophylactic and therapeutic vaccination in women with squamous intraepithelial lesions and with persistence of infection will be analyzed secondary end point
The investigators will analyze the patients enrolled prospectively with abnormal pap smear every six months up to two years after diagnosis At each time point during colposcopy peripheral blood 30 mL and cervical samples vaginal washing cervical biopsy and pap-smear will be collected for the characterization of local and peripheral immune response Viral load HPV integration will be assessed on cytological and tissue samples obtained from the patients at time of diagnosis andor curative surgery
For the secondary endpoints the investigators will enroll patients with confirmed HPV infection undergoing therapeutic and prophylactic HPV vaccination The vaccination will be performed immediately after HPV lesion diagnosis Follow-up program implies colposcopy every six months for 2 years The investigators will analyze HPV-specific immune response every six months and viral load and HPV integration at the time of diagnosis and at the end of the follow-up
To evaluate antigen-specific proliferative response PBMCs will be stimulated in triplicate in 96-well round-bottom plates with antigens L1 E6 E7 proteins of HPV- 16 and -18 and human Actin peptide pools for 7 days Culture medium was RPMI 1640 supplemented with 2 mM L-glutamine 100 UmL penicillin and 100 µgmL streptomycin solution 10 of heat inactivated FBS 1 mM Sodium Pyruvate 100 µM non-essential amino acids and 50 µM 2-Mercaptoethanol After culture cells will be washed stained with LiveDead Fixable Violet Dye and subsequently with CD3 CD4 CD8 CD25 CD278 ICOS Finally cells will be washed and resuspended in PBS 1 paraformaldehyde
A Cell Proliferation Index CPI for antigen-specific expanded T-cells will be determined by subtracting the percentage of CD25ICOS CD3CD4 or CD3CD8 detected in PBMC incubated with actin peptides from the percentage of CD25ICOS T-cell subsets detected in PBMC incubated with HPV peptides A CPI 15 will be considered negative while a value 15 will be considered positive
Flow cytometry analyses were performed with a FACS Canto II flow cytometer and BD DIVA software
For the analysis of HPV-related features molecular approaches will be used HPV DNA will be extracted from biopsies and lesion swabs using automatized systems after specific lysis treatment for tissues
HPV DNA will be then quantified using real time PCR specific for L1E2 and E6 regions in detail MY 0911 fragment of L1 gene will be used Results will be expressed as the number of copies100000 cells and normalized using a housekeeping gene
For the differential quantification of integrated or episomal HPV DNA a specific standardized system for the amplification of ORF E2 and E6 will be used
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None