Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT06339684 |
| Other study ID # |
2021 0075257 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
March 8, 2023 |
| Est. completion date |
March 8, 2026 |
Study information
| Verified date |
April 2024 |
| Source |
Foundation IRCCS San Matteo Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational [Patient Registry]
|
Clinical Trial 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.
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 and/or 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 U/mL penicillin and 100 µg/mL 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 Live/Dead 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 CD25+ICOS+ CD3+CD4+ or CD3+CD8+ detected in PBMC incubated with
actin peptides from the percentage of CD25+ICOS+ T-cell subsets detected in PBMC incubated
with HPV peptides. A CPI <1.5% will be considered negative while a value ≥1.5% 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 L1/E2 and E6 regions; in
detail MY 09/11 fragment of L1 gene will be used. Results will be expressed as the number of
copies/100.000 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.
