Immunohistochemical Evaluation of Protein P16 Expression in Ovarian Germ Cell Tumors.
Condition: Ovarian Neoplasms; Germ Cell Tumors; Immunohistochemistry
MeSH terms: Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms
Study type: Observational

Summary: 
      Ovarian germ cell tumors (OGCTs) constitute 10% of ovarian tumors in Egypt and mainly affect
      young females. Teratomas are the most common type.Most of teratomas is benign. However, it is
      liable for malignant transformation. Others are malignant including dysgerminoma, immature
      teratoma, yolk sac tumor,.etc and accounts 1-1.5% of cancers in young females. The
      pathogenesis of OGCTs is not clearly understood.

      P16 is a member of cyclin-dependent kinase (CDK) inhibitors. It arrests the cell cycle in G1
      phase, so it is known as a tumor suppressor protein.P16 immunohistochemical(IHC) expression
      has been widely investigated in different cancers. Its IHC expression is either absent or
      overexpressed. Overexpression of p16 is documented in Human Papilloma Virus related
      endocervical neoplasms and High grade squamous intraepithelial lesions of the vulvovaginal
      region.Absence of p16 expression is detected in multiple cancers such as Lung cancer,
      colorectal cancer and lymphoma.

      P16 IHC expression in OGCTs is poorly investigated. One study suggests that absent p16 is
      involved in proliferation of malignant OGCTs via molecular assessment.Another study suggested
      that decrease P16 is involved in malignant transformation of Mature cystic teratoma to
      squamous cell carcinoma.However, Previous studies are still limited and recommended further
      studies to confirm its results.

      As the role of altered P16 protein in OGCTs is not widely investigated, we hypothesized that
      abnormal P16 expression may be involved in its pathogenesis and germ stem cell
      proliferation.This will give more information about molecular pathways of germ stem cell
      proliferation to give a hope for CDK inhibitors as novel target therapies in the management
      of OGCTs.
    

Detailed description: 
      In this study , we aim to :

      1-Evaluate the immunohistochemical expression of p16 in neoplastic component of benign and
      malignant OGCTs and scoring its immunoreactivity.

      2-Correlate between P16 immunohistochemical expression in MOGCTs with Ki 67 proliferating
      index and FIGO staging.

        1. Type of the study: Matched case-control study.

        2. Study subjects:

             1. Inclusion criteria: Different types of Ovarian germ cell tumors including : benign
                ( mature cystic teratoma ) and malignant ones ( dysgerminoma , immature teratoma
                and yolk sac tumor ) .

             2. Exclusion criteria:

                Any criteria that not fulfill the inclusion criteria such as resected ovarian
                tumors other than ovarian germ cell tumors e.g epithelial ovarian tumors, sex cord
                -stromal tumors or metastatic ovarian lesions.

                  -  The study protocol was approved by the Institutional Review Board at Faculty
                     of medicine, Assiut University in December, 2019 (IRB No.17100871) and Ethical
                     approval for this study was obtained from the committee of medical ethics,
                     Faculty of medicine, Assiut University.

                  -  Study Setting: IHC lab of Pathology department, Faculty of medicine, Assiut
                     University.

                  -  Tissue sampling: Sixty-two formalin fixed ,paraffin wax-embedded ovarian
                     specimens were obtained from departments of surgical pathology in Assiut
                     University Hospital (AUH) and South Egypt cancer institute (SECI) from January
                     2010 to January 2021. These specimens includes:

                (Group A) 22 malignant ovarian germ cell tumors (MOGCTs), (Group B) 20 apparent
                normal ovarian tissue as a control group and (Group C) 20 mature cystic teratomas
                as equal benign comparison group.

                Group A constitutes 5 dysgerminomas, 8 immature teratomas (four of them Grade II
                and the other four are Grade III, FIGO grading system) and 9 yolk sac tumors. The
                patients ranged in age from 12 to 23 years (median age 16.5 years). The initial
                diagnosis of each MOGCTs was re-evaluated according to the WHO Classification of
                Ovarian Tumors.

                Group B includes normally apparent ovaries that surgically resected with specimens
                of total abdominal hysterectomy and salpingo-opherctomy for non-ovarian,
                non-malignant causes as multiple fibroid uterus and adenomyosis as a control group.
                The patients ranged in age from 42 to 64 years (median 50 years old).

                All available Hematoxylin and eosin stained slides were examined by two independent
                pathologists by routine light microscopy and the most representative one or two
                tissue blocks were used for immunohistochemical staining. The clinical and
                pathological data includes age, grade, stage, recurrence of lesions were evaluated
                according to pathological reports.

                  -  Immunohistochemistry: The tissue samples were formalin-fixed, paraffin
                     embedded tissue blocks, and were stored at room temperature. Thin sections of
                     4 um thickness were cut from selected representative tissue blocks, and were
                     taken on to coated slides, kept in an oven at 70-C for 25 minutes,
                     deparaffinized by washing in two containers of xylene for 15 minutes for each
                     and rehydrated through serial dilutions (100% , 90% , 80% , 70 %) of alcohol
                     for 5 minutes in each dilution. Then, sections were treated by 3% Hydrogen
                     peroxide for 10 minutes to prevent nonspecific background followed by PBS
                     buffer wash 4 times adequately. For antigen retrieval, the slides were kept in
                     citrate buffer (PH = 6) and autoclaved at 90-C for 15 minutes and left to cool
                     to room temperature. The slides were washed with PBS buffer 4 times adequately
                     and the tissue sections were incubated with primary antibody; p16INK4a
                     Recombinant Rabbit Monoclonal Antibody (RM267) (MA5-27905) at a dilution of
                     1:500 overnight, followed by incubation with secondary antibody; Polyvalent
                     polymer-HRP for 10 minutes for each step followed by adequate 4 times of PBS
                     buffer wash. The tissues were then treated with Diaminobenzidine chromogen
                     (DAP-chromogen) for brown color development for 10 minutes. Then, sections
                     were treated by Mayer hematoxylin as the counter stain for 2 minutes.

                For sections received from malignant cases only , we add additional Ki67 IHC
                staining as tissue sections after deparffinization and blocking by 3% hydrogen
                peroxidase were incubated with primary antibody; Ki67 antibody (DAKO), ready to use
                for 20 minutes, followed by incubation with secondary antibody; Polyvalent
                polymer-HRP for 20 minutes, followed treatment with Diaminobenzidine chromogen
                (DAP-chromogen) for brown color development for 5 minutes. Then, sections were
                treated by Mayer hematoxylin as the counter stain for 2 minutes.

                The slides were washed by tap water, dehydrated through serial dilutions of
                alcohol, washed by xylene for a short period, and mounted with DPX.

                The normal apparent ovarian tissues were used as controls. Sections from cervical
                non keratinized Squamous cell carcinoma with block p16 expression, were used as
                positive controls; and the tissue slides without the primary antibody treatment
                served as the negative controls.

                -Immunohistochemical evaluation: For P16 IHC staining, the percentage of P16
                positive cells and the location of positive signals (nuclear or cytoplasmic) were
                visually estimated for neoplastic components of all lesions. German
                Semi-quantitative scoring system were used to evaluate P16 expression as every
                tumor will be given a score according to the intensity of the cytoplasmic and
                nucleic staining (no staining = 0, weak staining = 1, moderate staining = 2, strong
                staining = 3) And the extent of stained cells (0% = 0, 1-10% = 1, 11-50% =2, 51-80%
                = 3, 81-100% = 4). The final immunoreactive score will be determined by multiplying
                the intensity scores with the extent of positivity scores of stained cells, with
                the minimum score of 0 and a maximum score of 12 ( score 0, 1,2,3,4,6,8,9 and 12).

                For KI67 IHC staining for malignant cases only, percentage of nuclear positivity
                stained cells were assessed, regardless intensity of staining in all sections
                examined (at least 1000 tumor cells were counted per section for estimation of KI
                index).

                -Statistical analysis: Data were analysed using IBM-SPSS version 24. Numerical data
                were presented as mean and standard deviation, while categorical data were
                presented as number and percentage. Descriptive statistics: Means, standard
                deviations, medians, ranges, and percentages were calculated. Test of
                significances: for continuous variables with more than two categories; ANOVA test
                was calculated to test the mean differences of the data that follow normal
                distribution and independent sample Kruskal-Wallis was used to compare the median
                difference between groups that do not follow normal distribution, post-hoc test was
                calculated using Bonferroni corrections. Correlation analysis was used (Spearman'
                Ranked correlation, 2-tailed).

                A p-value < 0.05 was considered significant.
    

Intervention name(s): p16INK4a Recombinant Rabbit Monoclonal Antibody (RM267); Ki67 antibody (DAKO) for malignant cases only
Intervention type(s): Combination Product; Combination Product
Intervention description(s): Treatment of slides that cut from paraffin embedded blocks related to groups by immunohistochemical method by p16INK4a Recombinant Rabbit Monoclonal Antibody (RM267).; Treatment of slides that cut from paraffin embedded blocks related to malignant case group by immunohistochemical method by Ki67 antibody.
Intervention MeSH terms: Antibodies; Immunoglobulins; Antibodies, Monoclonal
Observational model: Case-Control
Time perspective: Retrospective
Sampling method: Probability Sample
Study population: 
        Sixty-two formalin-fixed, paraffin wax-embedded ovarian specimens .These specimens
        includes:

        (Group A) 22 malignant ovarian germ cell tumors (MOGCTs): 5 dysgerminomas, 8 immature
        teratomas (four of them Grade II and the other four are Grade III, FIGO grading system) and
        9 yolk sac tumors. The patients ranged in age from 12 to 23 years (median age 16.5 years).
        The initial diagnosis was re-evaluated according to the WHO Classification of Ovarian
        Tumors.

        , (Group B) 20 apparent normal ovarian tissue as a control group that are surgically
        resected with TAH and salpingo-opherctomy for non-ovarian, non-malignant causes. The
        patients ranged in age from 42 to 64 years (median 50 years old).

        and(Group C) 20 mature cystic teratomas as equal benign comparison group.
      
Biospecimen description: 
      Formalin fixed paraffin embedded blocks of 62 ovarian specimens will be included in this
      study. These specimens will be divided into 2 groups:

      1-42 surgically resected ovarian germ cell tumors specimens: 22 malignant ones( dysgerminoma
      , immature teratoma and yolk sac tumor) as a case group and equal 20 benign ones (mature
      cystic teratoma ) as comparison group .

      2- 20 normally apparent ovaries that surgically resected with specimens of total abdominal
      hysterectomy and salpingo-Oopherctomy for non-ovarian causes in perimenopausal women as a
      control group .
    
Keywords: OGCTs, P16 , Ki67 , IHC