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

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    Assiut University
Updated on 15 September 2021
breast cancer
germ cell tumor


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:
  3. Inclusion criteria: Different types of Ovarian germ cell tumors including : benign ( mature cystic teratoma ) and malignant ones ( dysgerminoma , immature teratoma and yolk sac tumor ) .
  4. 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.

Condition Ovarian disorder, Ovarian Cancer, Ovarian Function, Recurrent Ovarian Cancer, ovarian tumors
Treatment Immunohistochemistry
Clinical Study IdentifierNCT04283773
SponsorAssiut University
Last Modified on15 September 2021

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