[version 1; peer review: awaiting peer review]
No competing interests were disclosed.
Universally, ovarian carcinoma (OC) is the deadliest gynecological tumor. It is ranted as the fifth most prevalent disease causing mortality in women
The phosphatidylinositol-3-kinases (PI3Ks) are a member of the kinase family
In OC, PIK3CA gene mutation or amplification is present in >20% of women
This hospital-based, cross-sectional study included 90 adults patients (convenience sampling), out of which, 83 patients diagnosed with OC at Omdurman Maternity Hospital in the period 2013–2018. Another seven healthy ovarian tissues (taken from hospital records of cases diagnosed as unremarkable, which means no histological change) were used as controls to normalize the results. All cases were adults with no history for previous malignancies. The data was collected in the period between 2016 and 2018.
The Ministry of Health of Khartoum state, AlzaiemAlazhari University, and Omdurman Maternity Hospital Ethical Committees approved the study. Informed consent from patients was waived by the committees, since patients’ identity was anonymized, and only laboratory numbers were used.
Total RNA was extracted from the FFPE tissue sections using the total RNeasy FFPE kit from Qiagen© (Qiagen, Hilden, Germany), according to the manufacturer’s guidelines. To synthesize cDNA, QuantiTect Reverse Transcription Kit (Qiagen) was used according to the manufacturer’s information. The cDNA was stored at -20°C until use.
Amplification of the PIK3CA gene was assessed using quantitative real-time PCR (qPCR), performed in ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the QuantiTectSYBR Green PCR Kit (Qiagen). DNA content was standardized to that of long interspersed elements (LINE1), which is a repetitive element with similar copy number per haploid genome, both in normal DNA samples and cancer cells
| PIK3CA | F, 5'-TATGGTTGTC TGTCAATCGGTGA-3' |
| R,5'-GCCTTTGCAGTGAATT-TGCAT-3' | |
| LINE1 | F, 5'CCGCTCAACTACATGGAAACTG-3' |
| R, 5'GCGTCCCAGAGATTCTGGTATG-3' |
The whole reaction volume of 20μl consisted of 10μl of SYBR Green Master Mix, 1μl forward and reverse primers (Macrogen, Korea), 1 μl cDNA, and RNA free water to complete the volume to 20μl. The following cycling conditions were used: 95°C for ten mins; 50 cycles with an annealing temperature of 55°C for 30s; and ending with an extension step at 72°C for 30s for both PIK3CA and LINE1 primers. A negative control (nuclease-free water as an alternative of cDNA) was incorporated in each run. A melting curve was obtained to provide evidence for a single reaction product. The relative quantification for PIK3CA gene was calculated using the 2
- ΔΔCt method
The results were analyzed using SPSS, version 24 (IBM SPSS). Descriptive analysis was performed for all variables (tumor type, grade, and age of women), which were taken from patient records. Fisher’s exact test was used between PIK3CA gene amplification and tumor types, grades, and ages for finding statistical significance. P-value < 0.05 was considered to be statistically significant.
Missing data in the dataset (
This study included 90 ovarian samples. Among the 90 cases, the median age was 50 years (range, 18–75 years). All cases were distributed into two age groups: ≤ 50 years (range, 18–50 years), 48.2% (n = 40); > 50 years (range, 51–75 years), 38.6% (n = 32).
Amplification of the PIK3CA was common in 33.7% of OC samples (n = 28/83). The results of the amplification rate in different OC types is presented in
The phosphatidylinositol-3-kinase (PI3K) pathway plays a regulatory role in common physiological processes; when mutated, it contributes to cellular tumorigenesis, cancer evolution, and drug conflict
Regarding the association of amplification with tumor grade; we observed an increased amplification frequency in higher grade carcinoma compared with low-grade carcinoma. Our observation implies that PIK3CA gene amplification is an early result in the progress of poorly differentiated (high grade) OC. However, amplification was not significantly associated with tumor type or grade in the present study. Our results are supportive of previous studies
The small sample size limited our study. Further studies with an increased sample size and more additional data (e.g. stage at diagnosis, chemotherapy, radiation) are expected to verify the outcome of this study. The significance of combining other genes, which exist in the PIK/AKT pathway, should also be investigated.
The present study revealed that amplification of the PIK3CA gene occurs in about one-third of Sudanese women with OC, more frequently in high grade carcinoma and women of older ages. In support of recently published literature, our observations foster early reports that imply that oncogenic PIK3CA gene has a significant role in the progression and activation of OC and might offer a new strategy for specific targeted therapy and prognostic assessment in OC patients.
Figshare: pik3ca data.csv,
Data are available under the terms of the
We are grateful to the staff at Omdurman Maternity Hospital (Department of Histopathology and Cytology) for their support. The authors express gratitude to the Institute of Endemic Diseases, University of Khartoum, Sudan, and Color-Lab (Qiagen), Egypt.