Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT06041464 |
| Other study ID # |
3740 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
March 2, 2021 |
| Est. completion date |
March 2, 2024 |
Study information
| Verified date |
September 2022 |
| Source |
Fondazione Policlinico Universitario Agostino Gemelli IRCCS |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
In our previous study (title: Expression of Major Histocompatibility Complex Molecules class
II- HLA-DR in Squamous Cell Carcinoma of the Head and Neck. ID 2222)the Authors verified that
the epithelial cells of squamous cell carcinoma of the head and neck acquire the ability to
express HLA-DR. Although the role of the expression of these molecules on neoplastic cells
still remains controversial, a positive association between HLA-DR expression and clinical
outcome was observed by us in analogy to what was reported by several studies on various
types of tumors : in squamous cell carcinoma of the larynx, colorectal cancer , stomach
cancer and others. In these tumors the expression of HLA-DR correlates with the presence of
immune cells such as CD16+/CD11c macrophage myeloid cells, associated with a good prognosis
and T cells which, recalled in the damaged tissue, they determine the formation of an
immunogenic microenvironment that could support an anti-tumor immune response. Oncology
studies are in fact focusing on the role of the tumor microenvironment which is characterized
by different cell populations, among which the most abundant population is represented by
tumor-associated fibroblasts (CAF). CAFs are fibroblasts which, in a tumor context, assume a
phenotype similar to that of myo-fibroblasts and are distinguishable from normal fibroblasts
by a greater expression of α-sma, FAP and FSP-1, which represent their specific markers, as
well as a greater expression of vimentin, fibronectin, and type XI collagen. Numerous
evidences in different types of tumors have reported both the immunosuppressive role, as
these cells are capable in vitro of inhibiting the proliferation of T lymphocytes, to favor
their apoptosis or to induce the phenotype of regulatory T lymphocytes; and the pro-tumor
role, as they are capable of promoting tumor proliferation and invasion, angiogenesis and
metastasis, thus contributing to the worsening of the prognosis. Many studies are directing
their research on which factors secreted by CAFs are responsible for their function. In
particular, among the many factors secreted by CAFs, there are the interleukins IL-17 and
IL-33 which, as it has been demonstrated, can induce the activation of HLA-DR molecules on
bone marrow-derived mesenchymal cells . It therefore seems interesting to investigate the
role of HLA-DR in relation to the presence of the tumor microenvironment represented by CAFs.
Description:
Several studies have demonstrated a correlation between the expression on tumor cells of MHC
II (major histocompatibility complex class II) molecules, in particular of HLA-DR (human
luekocyte antigen) molecules, and the clinical outcome of the disease. Indeed, it has been
observed that tumor cells under inflammatory stimulation of IFN-γ become capable of
expressing MHC II molecules functioning as non-professional APCs (antigen presenting cells).
The APC cells are responsible for presenting the antigen to the T lymphocytes, thus inducing
their activation, which necessarily requires the presence of two signals: the first induced
by the MHC molecules (MHC class II for CD4+ lymphocytes) and the second induced by
Costimulatory molecules CD80 or CD86, without which lymphocytes undergo a state of anergy.
Although the role of the expression of these molecules on neoplastic cells still remains
highly controversial, a positive association between HLA-DR expression and clinical outcome
has been reported by several studies on various types of tumors: in squamous cell carcinoma
of the larynx , colorectal cancer , stomach cancer , and others. Also in our study
"Expression of the Molecules of the Major Histocompatibility Complex class II-HLA-DR in
Squamous Cell Carcinoma of the Head and Neck" (ID 2222) the investigators were able to verify
that the neoplastic epithelial cells of squamous cell carcinoma of the head and neck acquire
the ability to express HLA-DR (data not shown, in progress of the scientific work to be
presented to journal with impact factor and of the final PhD thesis in Neuroscience of the
PhD student Chiara Prampolini). In some types of tumors, the expression of HLA-DR correlates
with the presence of immune cells such as CD16+/CD11c macrophage myeloid cells, associated
with a good prognosis and T cells which, recalled in damaged tissue, thus determine the
formation of an immunogenic microenvironment which could thus support an anti-tumor immune
response . Therefore, the variability in the expression of HLA-DR molecules would explain the
different immune involvement in the tumor site and would condition the host's ability to stem
tumor progression.
A fundamental role in tumor progression is the microenvironment, on which more and more
studies are focusing. The tumor microenvironment is characterized by different cell
populations, among which the most abundant population is represented by tumor-associated
fibroblasts (CAF). CAFs are fibroblasts which, in a tumor context, assume a phenotype similar
to that of myo-fibroblasts and are distinguishable from normal fibroblasts by a greater
expression of α-sma, FAP and FSP-1, which represent their specific markers, as well as a
greater expression of vimentin, fibronectin, and type XI collagen. Numerous evidences in
different types of tumors have reported both the immunosuppressive role, as these cells are
capable in vitro of inhibiting the proliferation of T lymphocytes, to favor their apoptosis
or to induce the phenotype of regulatory T lymphocytes; and the pro-tumor role, as they are
capable of promoting tumor proliferation and invasion, angiogenesis and metastasis, thus
contributing to the worsening of the prognosis.
Many studies are directing their research on which factors secreted by CAFs are responsible
for their function. In particular, among the many factors secreted by CAFs, there are the
interleukins IL-17 and IL-33 which, as it has been demonstrated, can induce the activation of
HLA-DR molecules on bone marrow-derived mesenchymal cells . It therefore seems interesting to
investigate the role of HLA-DR in relation to the presence of the tumor microenvironment
represented by CAFs. of the study OBJECTIVES OF THE STUDY Primary objective of the study
- Isolate tumor epithelial and cancer-associated fibroblast (CAF) cell cultures from
pathological and healthy tissue samples from patients with primary squamous cell
carcinoma of the head and neck Secondary objectives of the study
- To investigate the expression of HLA-DR in the obtained cell cultures. Enrolled patients
and enrollment methods Calculation of the sample size The calculation of the sample size
was carried out using Power Analysis, deeming acceptable a first type error equal to 5%
with a test power of 90%, therefore a second type error of 10%. To determine the delta,
the minimum acceptable difference to be detected between the two groups being compared
(group A: healthy tissue; group B: pathological tissue) and the standard deviation
relating to the populations under examination, the calculations of the effect size were
followed guidelines proposed by Choen .
Patients Four patients with primary squamous cell carcinoma of the head and neck admitted for
surgical treatment at the Otolaryngology Clinic, Fondazione Policlinico Gemelli, IRCCS, Rome,
in the year 2020-2021 will be included in the study.
Of the 4 cases considered will be available:
- Clinical data
- Informed consent
- Tumor tissue sample
- Non-tumor tissue sample taken from material surgically removed during surgery
Informed consent The subjects involved in the study will be invited to read the project
information sheet and to personally sign the informed consent for the use of their stored
biological material and clinical data for research purposes.
Organic material For each patient included in the study, a biopsy will be taken both from the
pathological tissue and from the healthy tissue removed during the therapeutic surgery, used
as a control. Both biopsies will be cultured.
5. Study design The study is a non-profit, single-centre that consists of two phases. The
expected duration of the study is 12 months.
I PHASE:
Selection of cell populations
Through specific culture media will be selected:
- Epithelial cells from healthy biopsy and tumor epithelial cells from tumor biopsy
maintained in culture with: DMEM High Glucose + HAM'S F12 1:1, FBS 10%,
penicillin-streptomycin 1%, L-glutamine 1%, insulin 10µg /ml, EGF 20ng/ml,
Hydrocortisone 0.5µg/ml;
- Fibroblasts from healthy biopsy and tumor associated fibroblasts (CAF) from tumor biopsy
maintained in culture with: DMEM High Glucose, FBS 10%, penicillin-streptomycin 1%,
L-glutamine 1%. Both fibroblasts and CAFs will be isolated from epithelial and tumor
cell cultures, respectively, with a series of passages in trypsin-EDTA 0.25%
Cell populations from the healthy biopsy (healthy epithelial cells and fibroblasts) will be
used as negative control.
PHASE II: Evaluation of HLA-DR expression in tumor epithelial cells and tumor CAFs HLA-DR
positivity will be evaluated by double immunofluorescence analysis with the addition of an
epithelial marker (cytokeratin), in addition to the evaluation of the presence of classical
costimulatory molecules (CD80, CD86).
HLA-DR+ tumor cells that do not express the costimulatory molecules will be stimulated in
culture with the administration of IFN-ɣ and LPS, in order to induce their expression .
Statistic analysis Comparisons between categorical data (healthy and pathological tissue)
will be made by chi-square test or Fisher's exact test, where appropriate. All tests will be
two-tailed and an alpha <0.05 will be considered statistically significant.
