Immunohistochemical Evaluation of Protein P16 Expression in Ovarian Germ Cell Tumors.

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.

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.

Eligibility Criteria:
Inclusion Criteria:

* Ovarian germ cell tumors :

  1. 20 benign ( mature cystic teratoma ).
  2. 22 malignant ones ( 5 dysgerminoma , 8 immature teratoma and 9 yolk sac tumor).
  3. 20 Normal ovaries .

Exclusion Criteria:

1. Epithelial ovarian tumors
2. sex cord -stromal ovarian tumors.
3. metastatic ovarian lesions.