Immunohistochemical Evaluation of Protein P16 Expression in Ovarian Germ Cell Tumors.
Immunohistochemical Evaluation of Protein P16 Expression in Ovarian Germ Cell Tumors.
Ovarian germ cell tumors (OGCTs) constitute 10% of ovarian tumors in Egypt [1] and mainly
 affect young females [2].Teratomas are the most common type [3].Most of teratomas is benign.
 However, it is liable for malignant transformation [4]. Others are malignant including
 dysgerminoma, immature teratoma, yolk sac tumor,.etc.[5] and accounts 1-1.5% of cancers in
 young females [6]. The pathogenesis of OGCTs is not clearly understood. Some authors revealed
 that mature cystic teratoma is a form of human parthenogenesis [7], which makes important
 concerns about future of fertility and germ stem cell replacement therapy [8].

 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 [9]. CDK inhibitors now are evolving as
 novel target therapies under clinical trials such as Palbociclib, Abemaciclib and Ribociclib
 that offer a hope for different cancers, such as breast cancer [10] and non-small cell lung
 cancer [11].One member which is recently approved by US FDA is palbociclib for use in
 postmenopausal women with breast cancer [12].Thus, it is necessary to do further researches
 on its expression among different neoplasms.

 P16 immunohistochemical(IHC) expression has been widely investigated in different cancers.
 Its IHC expression is either absent [13] or overexpressed [14] .Overexpression of p16 is
 documented in Human Papilloma Virus related endocervical neoplasms and High grade squamous
 intraepithelial lesions of the vulvovaginal region [14].Absence of p16 expression is detected
 in multiple cancers such as Lung cancer, colorectal cancer and lymphoma [15].

 P16 IHC expression in OGCTs is poorly investigated. One study suggests that absent p16 is
 involved in proliferation of malignant OGCTs via molecular assessment[16].Another study
 suggested that decrease P16 is involved in malignant transformation of Mature cystic teratoma
 to squamous cell carcinoma [17].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.
 Abnormalities may be found either in neoplastic or stromal components. The role of stromal
 component of OGCTs as tumor microenvironment has no published studies yet.

 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 and new
 advances in use of germ stem cell replacement therapy.
In this study , we aim to :

 1-Evaluate the immunohistochemical expression of p16 in both neoplastic and stromal
 components of benign and malignant OGCTs and scoring its immunoreactivity.

 2-Correlate between P16 immunohistochemical expression with clinicopathological parameters
 (Age, Histopathological type, grading, and 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.

 3. Sample Size Calculation: by Open Epi version 3. As regards review of literature:
 Risk of exposure measures % of P16 protein IHC expression in tissue.

 In normal ovarian tissue as Control group: measured approximately (50%). In malignant
 OGCT as case group: has decreased or absent expression (equals 10 % or less).

 And benign OGCT will be represented as equal comparison group.

 3. Materials and methods :

 Formalin fixed paraffin embedded blocks of eighty ovarian specimens will be included in this
 study. These specimens will be divided into 2 groups:

 1. Forty surgically resected ovarian germ cell tumors specimens: twenty malignant ones(
 dysgerminoma , immature teratoma and yolk sac tumor) as a case group and equal twenty
 benign ones (mature cystic teratoma ) as comparison group with mean age 17 years old.

 2. Forty 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 with mean age 40 years old.

 Adjustment of confounders (as Age) is not to be statistically significant.

 These specimens will be retrieved from the surgical pathology Lab of Assiut University
 hospital (AUH) from January 2010 to January 2021.

 The clinical data includes age, marital status, parity and laterality of lesions will be
 evaluated according to pathological referral records.

 Histopathological examination of sections stained with hematoxylin and eosin from cases of
 ovarian germ cell tumors by routine light microscopy to determine type, grade and
 pathological stage of the tumor.

 Immunohistochemical stain using anti-p16 antibody with immunoperoxidase staining as a
 visualization method. Antibody dilution, antigen retrieval methods and incubation time will
 all be conducted according to the manufacturer's instructions.

 4- Scoring and evaluation of immunohistochemistry: Semi-quantitative approach using H-score
 will be used to evaluate the staining as previously mentioned. A final score ranges from
 0-300 will be obtained by multiplying the staining intensity (0: negative, 1: weakly
 positive, 2: moderately positive and 3: strongly positive) and the percentage of positive
 cells.

 5-Data management and analysis: Computer software: All statistical analysis will be performed
 using software package SPSS version 16.

 Statistical tests: The numerical data will be presented as mean and standard deviation, while
 categorical data will be presented as number and percentage. Chi-square test will be used to
 evaluate the association between P16 immunohistochemistry and pathological variables.
 Adjustment of confounders (as Age) is not to be statistically significant. P value less than
 0.05 will be considered statistically significant. Multivariate Cox regression analysis will
 be performed.
Inclusion Criteria:

 -  Ovarian germ cell tumors :

 1. benign ( mature cystic teratoma ).

 2. malignant ones ( dysgerminoma , immature teratoma and yolk sac tumor).

 Exclusion Criteria:

 1. Epithelial ovarian tumors

 2. sex cord -stromal ovarian tumors.

 3. metastatic ovarian lesions.