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Research Note

Pattern of triple negative epithelial ovarian cancer in indigenous African women

[version 1; peer review: 2 approved]
PUBLISHED 28 Sep 2016
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Abstract

Background: Triple negative epithelial ovarian cancer (TNEOC)  refers to ovarian carcinomas that do not express estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor- type 2 (HER-2/neu).  The aim of this study is to determine the pattern of triple negative epithelial ovarian cancer in indigenous African women.

Methods: We performed a retrospective review of ER, PR and HER-2/neu expression in 90 Nigerian patients with histologically diagnosed epithelial ovarian cancer. Lack of expression of ER, PR and HER2/neu antigens was used to determine carcinomas that are among the TNEOC. We also compared the clinicopathological parameters (age, International Federation of Gynaecology and Obstetrics (FIGO) stage, grade and histological subtype) in patients with TNEOC and non- TNEOC .

Results: Thirty-eight (42.2%) of the 90 tumours diagnosed as EOC were negative for ER, PR and HER2/neu expression. There was no significant association between TNEOC with other parameters such as age, FIGO stage and histological grade. Sixteen (66.7%) of the 24 mucinous carcinomas were triple negative, while only 21 (33.3%) of the 63 serous carcinomas were triple-negative and one (50%) of the two endometrioid carcinomas was triple negative. There was a significant association between triple-negative tumours and histological subtypes of EOC (p = 0.034).

Conclusions: A subtype of epithelial ovarian cancer that is negative for ER, PR and HER-2/neu has been discovered in indigenous African women. TNEOC expression is high and is comparable to the triple negative breast cancer subtype seen in people of African ancestry. Future study of TNEOC in a large sample size should be considered.

Keywords

Estrogen Receptor, HER-2/neu expression, Immunohistochemistry, Ovarian carcinoma, Progesterone Receptor, Triple negative

Introduction

Epithelial ovarian cancer (EOC) remains one of the leading causes of death in gynaecological malignancies in developed countries14. The initial symptoms of ovarian cancer are often ambiguous, therefore it goes undiagnosed until after the disease is far advanced and has spread throughout the abdomen or to distant organs5,6.

Steroid hormone receptors expression in epithelial ovarian cancers have been proposed to have therapeutic and prognostic relevance, as is the case in breast cancers7. The determination of tumour characteristics such as age, International Federation of Gynaecology and Obstetrics (FIGO) stage, grade and histological subtypes has been associated with clinical behaviour and impact on treatment and prognosis but have been found to be limited8. Among the biological parameters proposed as possible prognostic factors in ovarian cancer, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor- type 2 (HER-2/neu) have been tested as potential biomarkers that guide individualized treatment of the cancer5,6,9. Epithelial ovarian carcinoma results from repeated ovulations, where the cumulative effects of each minor trauma on the ovarian epithelium can lead to malignant transformation10. PR has been observed to predict better prognosis because of its protection against ovarian carcinoma development11,12. On the other hand, overexpression of ER has been found to be associated with poor prognosis due to its contribution to initiation and/or promotion of ovarian carcinogenesis10,13. The HER-2/neu has been shown to be over-expressed in approximately 20–30% of EOC with associated poor prognosis1416.

Triple negative epithelial ovarian cancer (TNEOC) cases have been found to be more aggressive and display a worse prognosis than non-TNEOC cases17. This was similarly observed in the studies of triple negative breast cancer18,19.

This study was designed to determine the pattern of TNEOC among indigenous African women and correlate it with clinicopathological parameters.

Methods

Patient selection

We performed a retrospective review of ER, PR and HER-2/neu expression in 90 patients with histologically diagnosed epithelial ovarian cancer seen at the University College Hospital, Ibadan, Nigeria between January 2006 and December 2012. Non-epithelial primary ovarian cancers and metastatic cancers in the ovary were not included in this study. The demographic data and clinical history of these cases were obtained from the case notes, surgical daybooks, surgical pathology request forms, post-mortem records and Cancer Registry data. Formalin-fixed paraffin-embedded tissue blocks of histologically diagnosed solid EOC between January 2006 and December 2012 were retrieved and used for the study. The microscopic grading (three-grade system) of Shimizu and Silverberg was used, which assesses architectural pattern, nuclear pleomorphism and mitotic activity20. All histological classification of the EOC was based on the 2013 World Health Organisation (WHO) classification of ovarian tumours21. The FIGO staging of the cases used for this study was extracted from the case notes of the patients.

Ethics

The ethical clearance for this study was obtained from the Joint University of Ibadan/University College Hospital Ethical Review Committee (approval number UI/EC/13/0050) according to the Declaration of Helsinki.

Immunohistochemistry

The immunostaining procedure for HER-2/neu was carried out in accordance with the previously published article22. For the immunostaining procedure, three sections each for ER, PR and HER-2/neu at 5µm were cut from each of the paraffin-embedded tissue blocks after deparaffinization in xylene (two aliquots for five minutes each with the xylene covering the slide entirely). The sections were then rehydrated in graded alcohol concentrations (two aliquots each of 100% and 95% each and a single aliquot of 70%) in 250ml couplin jars. The antibodies used were monoclonal mouse anti-human ERα (Dako USA; clone ID5) and monoclonal mouse anti-human PR (Dako USA; clone PgR636) which identify the ER and PR nuclear protein antigens. The primary antibody used for HER-2/neu antigen was polyclonal rabbit anti-human C-erbB-2 (MBO/TEG, Dako USA, 1:800). The tissue sections were immersed in EDTA buffer (pH 9.0) for ER, citrate buffer (pH 6.0) for PR and in 1M Tris buffer (pH 9.0) for HER-2/neu. These slides were then incubated at room temperature for 20 minutes with primary monoclonal antibodies against ER (Dako USA, clone 1D5; 1:50), PR (Dako USA, clone PgR636; 1:50) and polyclonal rabbit anti-human C-erbB-2 (MBO/TEG, Dako USA, 1:800) followed by incubation in biotin-labelled secondary antibodies, polyclonal goat anti-mouse antibody for both ER and PR, (Dako USA, REF: K0675, LOT: 10081219) and polyclonal goat anti-rabbit antibody for HER-2/neu, (kitR, K5001, Dako Denmark) for 20 minutes and streptavidin-peroxidase complex (Dako USA, REF: K0675, LOT: 10084687) for another twenty minutes. The antigen-antibody complex was precipitated with di-aminobenzidine (DAB) for light microscopy with DAB substrate and DAB chromogen in the ratio of 1ml to 1 drop respectively. This was thereafter counterstained in Mayer’s haematoxylin (Dako USA). Dehydration of the sections was performed in ascending grades of alcohol and cleared in xylene. The slides were coversliped with DPX mountant. Known cases of breast cancer with positive reactions for ER, PR and HER-2/neu were used as a positive control. Negative controls were cases of tumour sections that were pretreated in Tris but without primary antibody immunostaining. All slides were reviewed independently by the three of the authors and cases with discordant scores were re-evaluated to have a consensus score. Grading of nuclear ER and PR staining was performed using an immunoreactive H-scoring system {none= 0 (negative); 1–25%=1+ (weak); 26–50%=2+ (moderate); >50%=3+(strong)}11. HER-2/neu membrane staining was graded in according to the Hercep Test protocol system as 0, 1+, 2+ or 3+. Samples scored as 0 or 1+ were considered negative for HER-2/neu overexpression, 2+ was weakly positive and 3+ was strongly positive22,23. Photomicrographs of the specimens were taken using Olympus digital camera, DP 21 at 400X magnification (Figure 1).

3ef31bf7-d8ea-4988-ad79-55577b28502e_figure1.gif

Figure 1. Photomicrographs showing ER (left), PR (middle) and HER-2/neu (right) negative expression (TNEOC) (immunostaining, 400X).

Statistical analysis

The data obtained were subjected to statistical analysis using Statistical Package for Social Sciences (SPSS) version 20. Statistical analysis was used to evaluate statistical associations between TNEOC and clinicopathological parameters i.e. age, FIGO stage grade, and histological subtypes. Continuous variables were compared using the student’s T test and categorical variables were compared using the chi-square test, with the level of significance set at p <0.05.

Results

RAW DATA FOR PATTERN OF TRIPLE NEGATIVE EPITHELIAL OVARIAN CANCERS IN INDIGENOUS BLACK WOMEN
S/NOAGE/yearsBIOPSY SIDEHISTOLOGICAL DIAGNOSISFIGO StageHistological GradingEstrogen receptor (ER)Progesterone receptor (PR)HER-2/neu
155RightSEROUS CARCINOMAIIIInegativenegativenegative
231RightMUCINOUS CARCINOMAIIInegativenegativenegative
372BilateralSEROUS CARCINOMAIIIInegativeweakly positiveweakly positive
450RightSEROUS CARCINOMAIIIweakly positivestrongly positivestrongly positive
559RightSEROUS CARCINOMAIIIstrongly positivenegativenegative
660RightSEROUS CARCINOMAIIIInegativenegativestrongly positive
744LeftMUCINOUS CARCINOMAIIInegativenegativenegative
836RightSEROUS CARCINOMAIIIIIInegativenegativenegative
952BilateralSEROUS CARCINOMAIIIIIweakly positivestrongly positiveweakly positive
1075BilateralSEROUS CARCINOMAIIIIInegativenegativenegative
1165LeftSEROUS CARCINOMAIVIIIweakly positiveweakly positivestrongly positive
1248LeftMUCINOUS CARCINOMAIIInegativenegativeweakly positive
1354RightSEROUS CARCINOMAIIIIIweakly positivenegativenegative
1437RightMALIGNANT BRENNER TUMOURIIstrongly positivenegativestrongly positive
1561BilateralSEROUS CARCINOMAIIIIstrongly positiveweakly positiveweakly positive
1643RightSEROUS CARCINOMAIIInegativeweakly positivenegative
1741RightSEROUS CARCINOMAIVIIInegativenegativenegative
1849RightMUCINOUS CARCINOMAIIIIInegativenegativenegative
1940LeftMUCINOUS CARCINOMAIIIIInegativeweakly positivenegative
2050RightSEROUS CARCINOMAIVIIInegativenegativestrongly positive
2126LeftSEROUS CARCINOMAIIIIInegativenegativenegative
2245BilateralENDOMETRIOID CARCINOMAIIInegativenegativenegative
2330RightSEROUS CARCINOMAIVIIInegativenegativenegative
2447LeftSEROUS CARCINOMAIIIIIInegativeweakly positivenegative
2538LeftSEROUS CARCINOMAIIIIweakly positivestrongly positivenegative
2648LeftSEROUS CARCINOMAIIIIInegativestrongly positivestrongly positive
2745LeftSEROUS CARCINOMAIIIIweakly positiveweakly positivenegative
2870RightSEROUS CARCINOMAIIIIIweakly positivenegativenegative
2935BilateralMUCINOUS CARCINOMAIIIIIIweakly positivenegativenegative
3053RightMUCINOUS CARCINOMAIVIInegativenegativeweakly positive
3142BilateralSEROUS CARCINOMAIIIInegativenegativenegative
3275BilateralMUCINOUS CARCINOMAIIIInegativenegativenegative
3355BilateralSEROUS CARCINOMAIVInegativenegativenegative
3450BilateralSEROUS CARCINOMAIVIIIstrongly positivenegativenegative
3565BilateralSEROUS CARCINOMAIIIIInegativenegativenegative
3649RightSEROUS CARCINOMAIIIIIIstrongly positivesrongstrongly positive
3750RightSEROUS CARCINOMAIVIIInegativeweakly positivestrongly positive
3845LeftSEROUS CARCINOMAIIIIIIweakly positivestrongly positivestrongly positive
3952LeftSEROUS CARCINOMAIIIIweakly positivenegativenegative
4056RightSEROUS CARCINOMAIIIIInegativenegativenegative
4146BilateralMUCINOUS CARCINOMAIIIInegativestrongly positivenegative
4216BilateralMUCINOUS CARCINOMAIIIInegativenegativenegative
4355LeftMUCINOUS CARCINOMAIIInegativenegativenegative
4459BilateralSEROUS CARCINOMAIIIInegativenegativenegative
4569BilateralSEROUS CARCINOMAIIIIIIweakly positiveweakly positiveweakly positive
4647BilateralSEROUS CARCINOMAIIIIInegativenegativenegative
4782RightMUCINOUS CARCINOMAIIIIInegativenegativenegative
4844LeftMUCINOUS CARCINOMAIIInegativenegativenegative
4944RightENDOMETRIOID CARCINOMAIIIInegativestrongly positiveweakly positive
5060LeftSEROUS CARCINOMAIVIInegativenegativestrongly positive
5132RightMUCINOUS CARCINOMAIInegativenegativenegative
5259BilateralMUCINOUS CARCINOMAIIIInegativenegativenegative
5368RightSEROUS CARCINOMAIIIIIIweakly positiveweakly positiveweakly positive
5457LeftSEROUS CARCINOMAIIIIIInegativenegativeweakly positive
5536LeftMUCINOUS CARCINOMAIInegativenegativenegative
5649RightSEROUS CARCINOMAIInegativenegativestrongly positive
5739LeftMUCINOUS CARCINOMAIIIInegativenegativenegative
5865RightSEROUS CARCINOMAIIIInegativenegativenegative
5964LeftSEROUS CARCINOMAIVIIInegativeweakly positivenegative
6075BilateralMUCINOUS CARCINOMAIIIweakly positivenegativestrongly positive
6147BilateralMUCINOUS CARCINOMAIIIInegativenegativenegative
6251LeftSEROUS CARCINOMAIVIIInegativenegativenegative
6350RightMUCINOUS CARCINOMAIIIIIInegativenegativenegative
6428RightSEROUS CARCINOMAIIIIIInegativenegativeweakly positive
6553RightSEROUS CARCINOMAIVIIInegativenegativenegative
6646LeftSEROUS CARCINOMAIIIIIweakly positiveweakly positivenegative
6769LeftSEROUS CARCINOMAIVIIInegativeweakly positivenegative
6840RightSEROUS CARCINOMAIIIIIInegativeweakly positivenegative
6975RightSEROUS CARCINOMAIIIIInegativenegativestrongly positive
7052RightSEROUS CARCINOMAIIIIInegativenegativestrongly positive
7147BilateralSEROUS CARCINOMAIVIIInegativeweakly positiveweakly positive
7269BilateralMUCINOUS CARCINOMAIInegativeweakly positiveweakly positive
7356BilateralSEROUS CARCINOMAIIIInegativenegativenegative
7466RightSEROUS CARCINOMAIIIIInegativeweakly positivenegative
7540LeftSEROUS CARCINOMAIIIIInegativenegativenegative
7660RightSEROUS CARCINOMAIIIIInegativenegativenegative
7754BilateralSEROUS CARCINOMAIIIIIIweakly positivestrongly positivenegative
7854RightSEROUS CARCINOMAIVIInegativenegativeweakly positive
7958RightMUCINOUS CARCINOMAIIIInegativenegativenegative
8053BilateralSEROUS CARCINOMAIVIInegativenegativestrongly positive
8164BilateralSEROUS CARCINOMAIVIIIstrongly positivestrongly positivestrongly positive
8257RightSEROUS CARCINOMAIIIIIInegativenegativenegative
8354LeftMUCINOUS CARCINOMAIIInegativenegativestrongly positive
8442RightSEROUS CARCINOMAIIIIIInegativestrongly positiveweakly positive
8535LeftSEROUS CARCINOMAIIIInegativenegativenegative
8660BilateralSEROUS CARCINOMAIIIInegativenegativeweakly positive
8762BilateralSEROUS CARCINOMAIVIIInegativestrongly positivestrongly positive
8867BilateralSEROUS CARCINOMAIIIIInegativenegativenegative
8956BilateralMUCINOUS CARCINOMAIIIInegativenegativenegative
Dataset 1.Raw data for ‘Pattern of triple negative epithelial ovarian cancer in indigenous African women’.

Thirty-eight (42.2%) of the 90 epithelial ovarian cancers (EOC) were negative for ER, PR and HER-2/neu expression (Figure 1). There was no significant association between triple-negative EOC and age (p = 0.218), FIGO stage (p = 0.425) and histological grade (p= 0.269). There were more TNEOC cases seen in patients older than 40 years than those below 40 years of age. Of 38 cases of TNEOC, 21 (55.3%) were found in the early stage (FIGO stage I and II) of epithelial ovarian cancer and 17 (44.7%) were at the advanced stage.

However, sixteen (66.7%) of the 24 mucinous carcinomas were triple-negative, while only 21 (33.3%) of the 63 serous carcinomas were triple-negative and one (50%) of the two endometrioid carcinomas was triple-negative (Table 1). There was therefore a significant association between triple-negative tumours and histological subtypes of EOC (p = 0.034).

Table 1. Correlation between triple negative epithelial ovarian cancer and clinicopathological parameters.

TNEOCNon-TNEOCTotalP value
Age
<409413
>402948770.218
FIGO Stage
I-II212243
III-IV1730470.425
Histological grade
I10919
II161834
III122537
Histological subtypes
Serous Carcinoma214263
Mucinous Carcinoma16824
Endometrioid Carcinoma112
Malignant Brenner tumour0110.034
Total385290

Discussion

A subgroup of epithelial ovarian cancer that is negative for ER, PR and HER-2/neu expression has been identified among indigenous African women. This subgroup is known as triple negative epithelial ovarian cancer (TNEOC). According to ER, PR, and HER-2/neu expressions, a breast cancer subtype known as triple negative breast cancer (TNBC) has been identified24.

In our study, triple negative tumours accounted for 42.2% of EOC. This value contrasts with the results of other studies17,25 and compares with the results of previous study26. A significant percentage (66.7%) of mucinous carcinoma were negative for ER, PR and HER-2/neu and this was statistically significant (p=0.034). This finding contrasts what was found from previous studies where there was no significant association between the TNEOC and histological subtypes17,25,26. No significant association was also found between the TNEOC and histological grade unlike what was observed by Liu et al.17 and de Toledo et al.26 where TNEOC was significantly correlated with histological grade. There was no significant association between TNEOC and age and FIGO stage compared to the findings of other studies17,25,26.

Our findings were comparable with what was found by Huo et al. in the population differences in breast cancer where triple negativity was predominant (27%)19. In view of the fact that triple-negative breast cancers are more often seen in black Africans and African-Americans and are associated with a poorer prognosis than non-triple-negative breast cancers, further studies of TNEOC in different environments are required.

Conclusions

A subtype of epithelial ovarian cancer that is negative for ER, PR and HER-2/neu has been discovered in Nigeria. Its (TNEOC) expression is high and is comparable to the triple negative breast cancer subtype seen in people of African ancestry. Future study of TNEOC in a large sample size should be considered.

Data availability

F1000Research: Dataset 1. Raw data for ‘Pattern of triple negative epithelial ovarian cancer in indigenous African women’, 10.5256/f1000research.9632.d13677727

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Ajani MA, Salami AA, Awolude OA and Oluwasola AO. Pattern of triple negative epithelial ovarian cancer in indigenous African women [version 1; peer review: 2 approved]. F1000Research 2016, 5:2415 (https://doi.org/10.12688/f1000research.9632.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 13 Oct 2016
Saad Aliyu Ahmed, Department of Pathology, Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria 
Approved
VIEWS 9
  1. This is an interesting manuscript, well conceived and has addressed the objective of the research.
     
  2. The methodology is simple and reproducible.
     
  3. Result was clearly outlined and discussion
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Ahmed SA. Reviewer Report For: Pattern of triple negative epithelial ovarian cancer in indigenous African women [version 1; peer review: 2 approved]. F1000Research 2016, 5:2415 (https://doi.org/10.5256/f1000research.10379.r16697)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 11 Oct 2016
Akinwumi Komolafe, Department of Morbid Anatomy and Forensic Medicine, Obafemi Awolowo University, Ilé-Ifẹ̀, Nigeria 
Approved
VIEWS 9
I have gone through the whole paper and these are my comments:
  1. It is a well conceived and conducted research paper with the aims and objectives and rationale for the study well spelled out.
     
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Komolafe A. Reviewer Report For: Pattern of triple negative epithelial ovarian cancer in indigenous African women [version 1; peer review: 2 approved]. F1000Research 2016, 5:2415 (https://doi.org/10.5256/f1000research.10379.r16923)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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