Prognostic Value of Protein IMP3 Expression in Cervical Cancer
Condition: Cervical Carcinoma
MeSH terms: Uterine Cervical Neoplasms
Study type: Observational

Summary: 
      The results of recent research on cervical cancer and its precancerous lesions have linked
      the expression of IMP3 protein to cervical dysplasia and the possibility of severe cervical
      dysplasia (CIN III) progressing to squamous cell carcinoma. A higher expression of IMP3
      protein was found in the cytoplasms of severe cervical dysplasia (CIN III) cells and invasive
      tumor cells compared to CIN I and CIN II change cells. The sensitivity of IMP3 expression in
      tumor cells was 96%. In preparations that were IMP3 negative, no further monitoring and
      treatment revealed squamous cell carcinoma. Further analyzes indicated the possibility of
      determining IMP3 expression on first cervical biopsy specimens in patients with HSIL ( high
      grade squamous intraepithelial lesion) lesions as a biomarker to detect a subset of patients
      in whom lesion invasiveness can be expected.
    

Detailed description: 
      Molecular biomarkers could selectively identify patients with a more unfavorable clinical
      course. One of these biomarkers is IMP3 - insulin-like growth factor II mRNA binding protein.

      The IGF group is a complex regulatory network at the cellular and substantial levels and
      plays a key physiological role in the development of the organism and the maintenance of
      normal cellular function during fetal and postnatal life. Overexpression of growth factors
      and their receptors is a common event during carcinogenesis. The IGF signaling system is
      under strict control of the normal physiological state and a deviation in this delicate
      balance can be a trigger for many molecular events leading to the development of malignancy.
      One member of the IGF system is the insulin-like growth factor II mRNA binding protein (IMP)
      which has been found to be an activator of IGF-II translation mRNA thus gaining a dominant
      role in controlling IGF-II-dependent cell proliferation.

      The IMP protein family contains 3 proteins (IMP1, IMP2, IMP3). IMP3 is normally expressed in
      fetal tissues during embryogenesis. In adulthood, it appears as an oncoprotein that is
      expressed only in tumor tissues without the presence in the surrounding (adjacent) benign
      tissue. Previous in vitro studies have shown that IMP3 stimulates tumor cell proliferation by
      activating IGF II translation and adhesion and invasion via adhesion molecules and matrix
      metalloproteinases 1.

      The results of recent research on cervical cancer and its precancerous lesions have linked
      the expression of IMP3 protein to cervical dysplasia and the possibility of severe cervical
      dysplasia (CIN III) progressing to squamous cell carcinoma. A higher expression of IMP3
      protein was found in the cytoplasms of severe cervical dysplasia (CIN III) cells and invasive
      tumor cells compared to CIN I and CIN II change cells. The sensitivity of IMP3 expression in
      tumor cells was 96%. In preparations that were IMP3 negative, no further monitoring and
      treatment revealed squamous cell carcinoma. Further analyzes indicated the possibility of
      determining IMP3 expression on first cervical biopsy specimens in patients with HSIL lesions
      as a biomarker to detect a subset of patients in whom lesion invasiveness can be expected.

      Based on previous studies, a monoclonal antibody specific for IMP3 has been developed and is
      successfully used in routine immunohistochemical methods that study the expression of IMP3
      proteins in various malignancies. It has recently been found that the expression of IMP3
      protein is associated with a more aggressive clinical course and a less favorable outcome in
      both gynecological and malignant diseases of other organ systems.

      Research issues

      Despite previous knowledge of prevention, detection and treatment, cervical cancer still
      causes great pain and mortality. The clinical course has so far been overlooked with regard
      to the clinical and pathological stage of the disease, and based on histological parameters
      and anatomical extent of the disease, a decision was made on further treatment and prognosis
      of the disease.

      Previous research has only provided an introduction to the association of IMP3 protein
      expression with cervical cancer. An association was found between the increased expression of
      IMP3 protein in gynecological and other cancers in the body with a poorer disease outcome.

      This study would look at the expression of IMP3 protein in cervical squamous cell carcinoma
      with the aim of detecting and linking the increased expression of IMP3 protein in patients
      who are expected to have an unfavorable clinical course and a poorer prognosis. Only patients
      in FIGO 1A1 and 1B1 disease stages will be observed, which has not been done in previous
      studies. In this way, patients who would be treated surgically would be singled out, and in
      whom a worse clinical outcome could be predicted and additional treatment could be planned
      individually.

      Purpose of research

      The study will determine the importance of immunohistochemical expression of IMP3 protein in
      cervical squamous cell carcinoma as a prognostic factor associated with higher tumor
      histological grade, higher FIGO stage, poorer course and disease outcome.

      Reasearch hypothesis

      Immunohistochemical expression of IMP3 protein in cervical squamous cell carcinoma was
      associated with poorer clinical course and disease outcome, shorter disease-free interval
      duration, and poorer 5-year survival outcomes.

      Immunohistochemical determination of IMP3 protein expression in cervical cancer - as a new
      prognostic factor

      The following will be excluded from the research:

        -  patients who have undergone pre-operative chemo and / or radiotherapy

        -  patients with associated gynecological or other malignancies

        -  patients with other histological types of tumors (adenocarcinoma, adenosquamous,
           neuroendocrine, etc.) The research will be performed on pathohistological preparations
           of surgical material of patients operated for cervical cancer in SKB Mostar in the above
           period.

      The expression of IMP3 protein will be determined by immunohistochemical method and
      statistical analysis will be used to assess the association of expression with clinical and
      pathological disease progression and survival parameters.

      Insight into medical documentation (patient medical histories and medical documentation of
      oncology patients from the Department of Oncology and Gynecology Clinic SKB Mostar) will
      provide clinical data on the age of patients, clinical stage of the disease determined by
      FIGO classification criteria, recurrence / length of period without signs of disease and
      clinical outcome.

      Research methods:

      Tumor samples for analysis will be obtained by 4μ sections of archived paraffin blocks.

      Mouse monoclonal antihuman antibodies an IMP3 (DAKO) at a dilution of 1: 100 will be used for
      immunohistochemical analysis.

      The immunohistochemical process will begin by dewaxing the tissue first in xylene and then
      rehydrating in alcohols of ever lower concentrations. Incubation in 0.1% H2O2 (30 minutes at
      room temperature) inactivated endogenous peroxidase. In order to detect antigenic sites, the
      sections will be boiled in citrate buffer, pH 9, in a microwave oven at 95 ° C for 10
      minutes, then washed in phosphate buffered saline (PBS). After cooling to room temperature,
      the sections were incubated with primary antibodies for 60 minutes. After one hour of
      incubation with the primary antibody and washing in PBS, secondary detection with
      diaminobenzidine (DAB) will be used. An antibody-independent EnVision Detection Systems
      Peroxidase (DAB, Rabbit / Mouse K 5007) will be used for this purpose. After a 30-minute
      incubation and washing in PBS, the sections will be stained with diaminobenzidine (DAB) for
      10 minutes at room temperature and washed in distilled water, and stained with hemalaun. The
      incisions will then be fitted into the fitting medium and covered with coverslips. Cells that
      reacted with the appropriate primary antibodies should have stained cytoplasms.

      The results of immunohistochemical staining will be interpreted semiquantitatively. Samples
      with cytoplasmic staining in more than 10% of tumor cells will be considered as positive
      expression, and will be classified into 5 categories: 0 (1% -10% of stained cells), 1 (11%
      -25%), 2 (26% 50) %), 3 (51% -75%) and 4 (76% -100%). The intensity of IMP3 expression will
      be classified as negative, weak, moderate, or severe.

      Sections intended for negative control by immunohistochemical staining of each antigen will
      be subjected, along with other sections, to the same procedure, except that they will not
      incubate with the primary antibody, but will be in PBS during that time. Control slides by
      the manufacturer will be used as a positive control.

      The specimens will be examined with an Olympus BX40 light microscope (Olympus, Tokyo, Japan).

      Total patient survival (OS) was defined as the time period between the date of surgery and
      the date of the last clinical control or death from cervical cancer.

      Disease-free interval (DFS) is defined as the time interval between the date of surgery and
      the date of disease recurrence.

      Statistical data processing:

      The obtained research results will be stored in the MS Excel 2007 database, for all
      statistical analyzes the SPSS 17 statistical program will be used. Descriptive statistics
      methods will be used in data analysis. Nonparametric variables will be displayed as frequency
      and percentage. Depending on the data distribution, the parametric variables will be
      displayed as the arithmetic mean and standard deviation or as the median and interquartile
      range. The data distribution will be tested by the Kolmogorov-Smirnov test. A chi-square test
      and Fisher's exact test will be used to test for differences between groups for categorical
      variables where necessary. Student t-test and one-way analysis of variance (ANOVA) or
      Mann-Whitney U test and Kruskal-Wallis test as non-parametric alternatives in case the data
      distribution deviates from normal will be used to test the difference between parametric
      variables. Appropriate correlation and regression tests will be used to test the correlation
      between the examined variables. In the analysis of survival and disease recurrence, the
      Kaplan-Meier method and the Cox-Mantel log-rank test or the Mantel-Haenszel test will be used
      depending on the data obtained. The probability level of p <0.005 will be taken as
      statistically significant.
    

Intervention name(s): immunohistochemical analysis with murine monoclonal antihuman antibodies IMP3
Intervention type(s): Other
Intervention MeSH terms: Antibodies
Observational model: Cohort
Time perspective: Retrospective
Sampling method: Probability Sample
Study population: 
        Operated women in University Clinic Mostar, who were diagnosed with squamous cell carcinoma
        cervicis in period of January 2003 to Decembar 2013.
      
Biospecimen description: 
      Parts of cervix