xml: <clinical_study>
    <!-- This xml conforms to an XML Schema at:
      https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
    <required_header>
      <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
      <link_text>Link to the current ClinicalTrials.gov record.</link_text>
      <url>https://clinicaltrials.gov/ct2/show/NCT06041464</url>
    </required_header>
    <id_info>
      <org_study_id>3740</org_study_id>
      <nct_id>NCT06041464</nct_id>
    </id_info>
    <brief_title>Study of HLA-DR in Cell Cultures Isolated From Primary Squamous Cell Carcinoma</brief_title>
    <official_title>Isolation of Cultures of Epithelial Tumor Cells and Cancer-associated Fibroblasts (CAF) From Primary Squamous Cell Carcinoma of the Head and Neck and Study of HLA-DR Expression</official_title>
    <sponsors>
      <lead_sponsor>
        <agency>Fondazione Policlinico Universitario Agostino Gemelli IRCCS</agency>
        <agency_class>Other</agency_class>
      </lead_sponsor>
    </sponsors>
    <source>Fondazione Policlinico Universitario Agostino Gemelli IRCCS</source>
    <oversight_info>
      <has_dmc>No</has_dmc>
      <is_fda_regulated_drug>No</is_fda_regulated_drug>
      <is_fda_regulated_device>No</is_fda_regulated_device>
    </oversight_info>
    <brief_summary>
      <textblock>
        In our previous study (title: Expression of Major Histocompatibility Complex Molecules class&#xD;
        II- HLA-DR in Squamous Cell Carcinoma of the Head and Neck. ID 2222)the Authors verified that&#xD;
        the epithelial cells of squamous cell carcinoma of the head and neck acquire the ability to&#xD;
        express HLA-DR. Although the role of the expression of these molecules on neoplastic cells&#xD;
        still remains controversial, a positive association between HLA-DR expression and clinical&#xD;
        outcome was observed by us in analogy to what was reported by several studies on various&#xD;
        types of tumors : in squamous cell carcinoma of the larynx, colorectal cancer , stomach&#xD;
        cancer and others. In these tumors the expression of HLA-DR correlates with the presence of&#xD;
        immune cells such as CD16+/CD11c macrophage myeloid cells, associated with a good prognosis&#xD;
        and T cells which, recalled in the damaged tissue, they determine the formation of an&#xD;
        immunogenic microenvironment that could support an anti-tumor immune response. Oncology&#xD;
        studies are in fact focusing on the role of the tumor microenvironment which is characterized&#xD;
        by different cell populations, among which the most abundant population is represented by&#xD;
        tumor-associated fibroblasts (CAF). CAFs are fibroblasts which, in a tumor context, assume a&#xD;
        phenotype similar to that of myo-fibroblasts and are distinguishable from normal fibroblasts&#xD;
        by a greater expression of α-sma, FAP and FSP-1, which represent their specific markers, as&#xD;
        well as a greater expression of vimentin, fibronectin, and type XI collagen. Numerous&#xD;
        evidences in different types of tumors have reported both the immunosuppressive role, as&#xD;
        these cells are capable in vitro of inhibiting the proliferation of T lymphocytes, to favor&#xD;
        their apoptosis or to induce the phenotype of regulatory T lymphocytes; and the pro-tumor&#xD;
        role, as they are capable of promoting tumor proliferation and invasion, angiogenesis and&#xD;
        metastasis, thus contributing to the worsening of the prognosis. Many studies are directing&#xD;
        their research on which factors secreted by CAFs are responsible for their function. In&#xD;
        particular, among the many factors secreted by CAFs, there are the interleukins IL-17 and&#xD;
        IL-33 which, as it has been demonstrated, can induce the activation of HLA-DR molecules on&#xD;
        bone marrow-derived mesenchymal cells . It therefore seems interesting to investigate the&#xD;
        role of HLA-DR in relation to the presence of the tumor microenvironment represented by CAFs.&#xD;
      </textblock>
    </brief_summary>
    <detailed_description>
      <textblock>
        Several studies have demonstrated a correlation between the expression on tumor cells of MHC&#xD;
        II (major histocompatibility complex class II) molecules, in particular of HLA-DR (human&#xD;
        luekocyte antigen) molecules, and the clinical outcome of the disease. Indeed, it has been&#xD;
        observed that tumor cells under inflammatory stimulation of IFN-γ become capable of&#xD;
        expressing MHC II molecules functioning as non-professional APCs (antigen presenting cells).&#xD;
        The APC cells are responsible for presenting the antigen to the T lymphocytes, thus inducing&#xD;
        their activation, which necessarily requires the presence of two signals: the first induced&#xD;
        by the MHC molecules (MHC class II for CD4+ lymphocytes) and the second induced by&#xD;
        Costimulatory molecules CD80 or CD86, without which lymphocytes undergo a state of anergy.&#xD;
  &#xD;
        Although the role of the expression of these molecules on neoplastic cells still remains&#xD;
        highly controversial, a positive association between HLA-DR expression and clinical outcome&#xD;
        has been reported by several studies on various types of tumors: in squamous cell carcinoma&#xD;
        of the larynx , colorectal cancer , stomach cancer , and others. Also in our study&#xD;
        &quot;Expression of the Molecules of the Major Histocompatibility Complex class II-HLA-DR in&#xD;
        Squamous Cell Carcinoma of the Head and Neck&quot; (ID 2222) the investigators were able to verify&#xD;
        that the neoplastic epithelial cells of squamous cell carcinoma of the head and neck acquire&#xD;
        the ability to express HLA-DR (data not shown, in progress of the scientific work to be&#xD;
        presented to journal with impact factor and of the final PhD thesis in Neuroscience of the&#xD;
        PhD student Chiara Prampolini). In some types of tumors, the expression of HLA-DR correlates&#xD;
        with the presence of immune cells such as CD16+/CD11c macrophage myeloid cells, associated&#xD;
        with a good prognosis and T cells which, recalled in damaged tissue, thus determine the&#xD;
        formation of an immunogenic microenvironment which could thus support an anti-tumor immune&#xD;
        response . Therefore, the variability in the expression of HLA-DR molecules would explain the&#xD;
        different immune involvement in the tumor site and would condition the host's ability to stem&#xD;
        tumor progression.&#xD;
  &#xD;
        A fundamental role in tumor progression is the microenvironment, on which more and more&#xD;
        studies are focusing. The tumor microenvironment is characterized by different cell&#xD;
        populations, among which the most abundant population is represented by tumor-associated&#xD;
        fibroblasts (CAF). CAFs are fibroblasts which, in a tumor context, assume a phenotype similar&#xD;
        to that of myo-fibroblasts and are distinguishable from normal fibroblasts by a greater&#xD;
        expression of α-sma, FAP and FSP-1, which represent their specific markers, as well as a&#xD;
        greater expression of vimentin, fibronectin, and type XI collagen. Numerous evidences in&#xD;
        different types of tumors have reported both the immunosuppressive role, as these cells are&#xD;
        capable in vitro of inhibiting the proliferation of T lymphocytes, to favor their apoptosis&#xD;
        or to induce the phenotype of regulatory T lymphocytes; and the pro-tumor role, as they are&#xD;
        capable of promoting tumor proliferation and invasion, angiogenesis and metastasis, thus&#xD;
        contributing to the worsening of the prognosis.&#xD;
  &#xD;
        Many studies are directing their research on which factors secreted by CAFs are responsible&#xD;
        for their function. In particular, among the many factors secreted by CAFs, there are the&#xD;
        interleukins IL-17 and IL-33 which, as it has been demonstrated, can induce the activation of&#xD;
        HLA-DR molecules on bone marrow-derived mesenchymal cells . It therefore seems interesting to&#xD;
        investigate the role of HLA-DR in relation to the presence of the tumor microenvironment&#xD;
        represented by CAFs. of the study OBJECTIVES OF THE STUDY Primary objective of the study&#xD;
  &#xD;
          -  Isolate tumor epithelial and cancer-associated fibroblast (CAF) cell cultures from&#xD;
             pathological and healthy tissue samples from patients with primary squamous cell&#xD;
             carcinoma of the head and neck Secondary objectives of the study&#xD;
  &#xD;
          -  To investigate the expression of HLA-DR in the obtained cell cultures. Enrolled patients&#xD;
             and enrollment methods Calculation of the sample size The calculation of the sample size&#xD;
             was carried out using Power Analysis, deeming acceptable a first type error equal to 5%&#xD;
             with a test power of 90%, therefore a second type error of 10%. To determine the delta,&#xD;
             the minimum acceptable difference to be detected between the two groups being compared&#xD;
             (group A: healthy tissue; group B: pathological tissue) and the standard deviation&#xD;
             relating to the populations under examination, the calculations of the effect size were&#xD;
             followed guidelines proposed by Choen .&#xD;
  &#xD;
        Patients Four patients with primary squamous cell carcinoma of the head and neck admitted for&#xD;
        surgical treatment at the Otolaryngology Clinic, Fondazione Policlinico Gemelli, IRCCS, Rome,&#xD;
        in the year 2020-2021 will be included in the study.&#xD;
  &#xD;
        Of the 4 cases considered will be available:&#xD;
  &#xD;
          -  Clinical data&#xD;
  &#xD;
          -  Informed consent&#xD;
  &#xD;
          -  Tumor tissue sample&#xD;
  &#xD;
          -  Non-tumor tissue sample taken from material surgically removed during surgery&#xD;
  &#xD;
        Informed consent The subjects involved in the study will be invited to read the project&#xD;
        information sheet and to personally sign the informed consent for the use of their stored&#xD;
        biological material and clinical data for research purposes.&#xD;
  &#xD;
        Organic material For each patient included in the study, a biopsy will be taken both from the&#xD;
        pathological tissue and from the healthy tissue removed during the therapeutic surgery, used&#xD;
        as a control. Both biopsies will be cultured.&#xD;
  &#xD;
        5. Study design The study is a non-profit, single-centre that consists of two phases. The&#xD;
        expected duration of the study is 12 months.&#xD;
  &#xD;
        I PHASE:&#xD;
  &#xD;
        Selection of cell populations&#xD;
  &#xD;
        Through specific culture media will be selected:&#xD;
  &#xD;
          -  Epithelial cells from healthy biopsy and tumor epithelial cells from tumor biopsy&#xD;
             maintained in culture with: DMEM High Glucose + HAM'S F12 1:1, FBS 10%,&#xD;
             penicillin-streptomycin 1%, L-glutamine 1%, insulin 10µg /ml, EGF 20ng/ml,&#xD;
             Hydrocortisone 0.5µg/ml;&#xD;
  &#xD;
          -  Fibroblasts from healthy biopsy and tumor associated fibroblasts (CAF) from tumor biopsy&#xD;
             maintained in culture with: DMEM High Glucose, FBS 10%, penicillin-streptomycin 1%,&#xD;
             L-glutamine 1%. Both fibroblasts and CAFs will be isolated from epithelial and tumor&#xD;
             cell cultures, respectively, with a series of passages in trypsin-EDTA 0.25%&#xD;
  &#xD;
        Cell populations from the healthy biopsy (healthy epithelial cells and fibroblasts) will be&#xD;
        used as negative control.&#xD;
  &#xD;
        PHASE II: Evaluation of HLA-DR expression in tumor epithelial cells and tumor CAFs HLA-DR&#xD;
        positivity will be evaluated by double immunofluorescence analysis with the addition of an&#xD;
        epithelial marker (cytokeratin), in addition to the evaluation of the presence of classical&#xD;
        costimulatory molecules (CD80, CD86).&#xD;
  &#xD;
        HLA-DR+ tumor cells that do not express the costimulatory molecules will be stimulated in&#xD;
        culture with the administration of IFN-ɣ and LPS, in order to induce their expression .&#xD;
  &#xD;
        Statistic analysis Comparisons between categorical data (healthy and pathological tissue)&#xD;
        will be made by chi-square test or Fisher's exact test, where appropriate. All tests will be&#xD;
        two-tailed and an alpha &lt;0.05 will be considered statistically significant.&#xD;
      </textblock>
    </detailed_description>
    <overall_status>Active, not recruiting</overall_status>
    <start_date type="Actual">March 2, 2021</start_date>
    <completion_date type="Anticipated">March 2, 2024</completion_date>
    <primary_completion_date type="Anticipated">October 2, 2023</primary_completion_date>
    <study_type>Observational</study_type>
    <has_expanded_access>No</has_expanded_access>
    <study_design_info>
      <observational_model>Other</observational_model>
      <time_perspective>Other</time_perspective>
    </study_design_info>
    <primary_outcome>
      <measure>NUMBER OF FLASKS OF HUMAN HEALTHY TISSUE CELL CULTURE FROM THE HEALTHY HEALTHY BIOPSY BIOPSY</measure>
      <time_frame>FROM THE DATA OF HEALTHY EPITHELIAL CELLS AND FIBROBLASTS BIOPSY UNTIL THE DATE OF CELL COLTURE ISOLATION ASSESSED UP TO 4 WEEKS</time_frame>
    </primary_outcome>
    <primary_outcome>
      <measure>NUMBER OF FLASKS OF TUMOR ASSOCIATED EPITHELIAL CELL AND FIBROBLASTS ASSOCIATED CANCER CULTURE FROM TUMOR BIOPSY</measure>
      <time_frame>FROM THE DATA OF TUMOR EPITHELIAL CELLS AND FIBROBLASTS BIOPSY UNTIL THE DATE OF CELL COLTURE ISOLATION ASSESSED UP TO 4 WEEKS</time_frame>
    </primary_outcome>
    <secondary_outcome>
      <measure>ZERO UP TO 4 SCALE (NEGATIVE UP TO VERY INTENSIVE) IMMUNOHISTOCHEMICAL HLA-DR POSITIVITY IN HEALTHY EPITELIAL CELLS AND FIBROBLASTS CULTURES</measure>
      <time_frame>FROM LAST ENROLLED PATIENT CELL COLTURES ISOLATION UP TO 16 WEEKS</time_frame>
    </secondary_outcome>
    <secondary_outcome>
      <measure>ZERO UP TO 4 SCALE (NEGATIVE UP TO VERY INTENSIVE) IMMUNOHISTOCHEMICAL HLA-DR POSITIVITY IN TUMOR EPITELIAL CELLS AND FIBROBLASTS CULTURES</measure>
      <time_frame>FROM LAST ENROLLED PATIENT CELL COLTURES ISOLATION UP TO 16 WEEKS</time_frame>
    </secondary_outcome>
    <number_of_groups>2</number_of_groups>
    <enrollment type="Actual">4</enrollment>
    <condition>Squamous Cell Carcinoma of the Head and Neck</condition>
    <condition>Cancer</condition>
    <arm_group>
      <arm_group_label>cancer epithelial cell</arm_group_label>
      <description>cancer epithelial cells culture</description>
    </arm_group>
    <arm_group>
      <arm_group_label>cancer fibroblast culture</arm_group_label>
      <description>cancer fibroblast cells culture</description>
    </arm_group>
    <intervention>
      <intervention_type>Other</intervention_type>
      <intervention_name>cell culture</intervention_name>
      <description>isolation of cell culture</description>
      <arm_group_label>cancer epithelial cell</arm_group_label>
      <arm_group_label>cancer fibroblast culture</arm_group_label>
      <other_name>immunohistochemical detection</other_name>
    </intervention>
    <biospec_retention>Samples Without DNA</biospec_retention>
    <biospec_descr>
      <textblock>
        Epithelial cells from healthy biopsy and tumor epithelial cells from tumor biopsy maintained&#xD;
        in culture with: DMEM High Glucose + HAM'S F12 1:1, FBS 10%, penicillin-streptomycin 1%,&#xD;
        L-glutamine 1%, insulin 10µg /ml, EGF 20ng/ml, Hydrocortisone 0.5µg/ml;&#xD;
  &#xD;
        • Fibroblasts from healthy biopsy and tumor associated fibroblasts (CAF) from tumor biopsy&#xD;
        maintained in culture with: DMEM High Glucose, FBS 10%, penicillin-streptomycin 1%,&#xD;
        L-glutamine 1%. Both fibroblasts and CAFs will be isolated from epithelial and tumor cell&#xD;
        cultures, respectively, with a series of passages in trypsin-EDTA 0.25%&#xD;
      </textblock>
    </biospec_descr>
    <eligibility>
      <study_pop>
        <textblock>
          The calculation of the sample size was carried out using Power Analysis, deeming acceptable&#xD;
          a first type error equal to 5% with a test power of 90%, therefore a second type error of&#xD;
          10%.&#xD;
  &#xD;
          patients Four patients with primary squamous cell carcinoma of the head and neck admitted&#xD;
          for surgical treatment at the Otolaryngology Clinic, Fondazione Policlinico Gemelli, IRCCS,&#xD;
          Rome, in the year 2020-2021 will be included in the study.&#xD;
  &#xD;
          Of the 4 cases considered will be available:&#xD;
  &#xD;
            -  Clinical data&#xD;
  &#xD;
            -  Informed consent&#xD;
  &#xD;
            -  Tumor tissue sample&#xD;
  &#xD;
            -  Non-tumor tissue sample taken from material surgically removed during surgery&#xD;
        </textblock>
      </study_pop>
      <sampling_method>Non-Probability Sample</sampling_method>
      <criteria>
        <textblock>
          Inclusion Criteria:&#xD;
  &#xD;
            -  diagnosis of primary squamous cell carcinoma of the head and neck, 2) signing of&#xD;
               informed consent for study involvement.&#xD;
  &#xD;
          Exclusion Criteria:&#xD;
  &#xD;
          1) patients diagnosed with non-primary squamous cell carcinoma of the head and neck and&#xD;
          already undergoing radiotherapy, chemotherapy and immunotherapy, biological therapy.&#xD;
        </textblock>
      </criteria>
      <gender>All</gender>
      <minimum_age>N/A</minimum_age>
      <maximum_age>N/A</maximum_age>
      <healthy_volunteers>No</healthy_volunteers>
    </eligibility>
    <overall_official>
      <last_name>gabriella cadoni, Prof</last_name>
      <role>Principal Investigator</role>
      <affiliation>FONDAZIONE POLICLINICO UNIVERSITARIO GEMELLI, IRCCS</affiliation>
    </overall_official>
    <location>
      <facility>
        <name>Fondazione Policlinico Universitario Gemelli, Irccs</name>
        <address>
          <city>Roma</city>
          <state>Lazio</state>
          <zip>00168</zip>
          <country>Italy</country>
        </address>
      </facility>
    </location>
    <location_countries>
      <country>Italy</country>
    </location_countries>
    <verification_date>September 2022</verification_date>
    <study_first_submitted>February 7, 2023</study_first_submitted>
    <study_first_submitted_qc>September 11, 2023</study_first_submitted_qc>
    <study_first_posted type="Actual">September 18, 2023</study_first_posted>
    <last_update_submitted>September 11, 2023</last_update_submitted>
    <last_update_submitted_qc>September 11, 2023</last_update_submitted_qc>
    <last_update_posted type="Actual">September 18, 2023</last_update_posted>
    <responsible_party>
      <responsible_party_type>Sponsor</responsible_party_type>
    </responsible_party>
    <keyword>HEAD AND NECK</keyword>
    <keyword>CANCER</keyword>
    <keyword>IMMUNOLOGY</keyword>
    <condition_browse>
      <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
      <mesh_term>Carcinoma</mesh_term>
      <mesh_term>Carcinoma, Squamous Cell</mesh_term>
      <mesh_term>Squamous Cell Carcinoma of Head and Neck</mesh_term>
    </condition_browse>
    <patient_data>
      <sharing_ipd>No</sharing_ipd>
    </patient_data>
    <!-- Results have not yet been posted for this study                                          -->
  </clinical_study>
  
  
text: download date: ClinicalTrials.gov processed this data on September 20, 2023
  link text: Link to the current ClinicalTrials.gov record.
  url: https://clinicaltrials.gov/ct2/show/NCT06041464
  org study id: 3740
  nct id: NCT06041464
  lead sponsor: 
  has dmc: No
  is fda regulated drug: No
  is fda regulated device: No
  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.
  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.
  observational model: Other
  time perspective: Other
  measure: NUMBER OF FLASKS OF HUMAN HEALTHY TISSUE CELL CULTURE FROM THE HEALTHY HEALTHY BIOPSY BIOPSY
  time frame: FROM THE DATA OF HEALTHY EPITHELIAL CELLS AND FIBROBLASTS BIOPSY UNTIL THE DATE OF CELL COLTURE ISOLATION ASSESSED UP TO 4 WEEKS
  measure: NUMBER OF FLASKS OF TUMOR ASSOCIATED EPITHELIAL CELL AND FIBROBLASTS ASSOCIATED CANCER CULTURE FROM TUMOR BIOPSY
  time frame: FROM THE DATA OF TUMOR EPITHELIAL CELLS AND FIBROBLASTS BIOPSY UNTIL THE DATE OF CELL COLTURE ISOLATION ASSESSED UP TO 4 WEEKS
  measure: ZERO UP TO 4 SCALE (NEGATIVE UP TO VERY INTENSIVE) IMMUNOHISTOCHEMICAL HLA-DR POSITIVITY IN HEALTHY EPITELIAL CELLS AND FIBROBLASTS CULTURES
  time frame: FROM LAST ENROLLED PATIENT CELL COLTURES ISOLATION UP TO 16 WEEKS
  measure: ZERO UP TO 4 SCALE (NEGATIVE UP TO VERY INTENSIVE) IMMUNOHISTOCHEMICAL HLA-DR POSITIVITY IN TUMOR EPITELIAL CELLS AND FIBROBLASTS CULTURES
  time frame: FROM LAST ENROLLED PATIENT CELL COLTURES ISOLATION UP TO 16 WEEKS
  arm group label: cancer epithelial cell
  description: cancer epithelial cells culture
  arm group label: cancer fibroblast culture
  description: cancer fibroblast cells culture
  intervention type: Other
  intervention name: cell culture
  description: isolation of cell culture
  arm group label: cancer epithelial cell
  arm group label: cancer fibroblast culture
  other name: immunohistochemical detection
  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%
  study pop: 
  sampling method: Non-Probability Sample
  criteria: 
  gender: All
  minimum age: N/A
  maximum age: N/A
  healthy volunteers: No
  last name: gabriella cadoni, Prof
  role: Principal Investigator
  affiliation: FONDAZIONE POLICLINICO UNIVERSITARIO GEMELLI, IRCCS
  facility: 
  country: Italy
  responsible party type: Sponsor
  mesh term: Carcinoma
  mesh term: Carcinoma, Squamous Cell
  mesh term: Squamous Cell Carcinoma of Head and Neck
  sharing ipd: No