Cervical cancer is one of the most frequent cancer of women in 2012 representing 7.9% of all female cancers (27). Approximately 90% of the 270,000 deaths from cervical cancer in 2015 occurred in low and middle-income countries (27). This is the most common cause of female death by cancer in 43 countries. The estimated global economic burden of cervical cancer in 2009 was 3 billion US dollars.
Cancer causing infections, such as hepatitis and human papilloma virus (HPV), are responsible for up to 25% of cancer cases in low- and middle-income countries. Vaccination against these HPV and hepatitis B viruses could prevent 1 million cancer cases each year (28). HPV is recognized as the most important risk factor for cervical cancer for last 15 years (29). World health organization recognized sexually transmitted HPV infection as a modifiable and avoidable risk factor.
Bangladesh has a population of 54.38 million women ages fifteen years and older who are at risk of developing cervical cancer. Current estimates indicate that every year 11,956 women are diagnosed with cervical cancer and 6,582 die from the disease (7). Cervical cancer ranks as the second most frequent cancer among women between 15 and 44 years of age in Bangladesh (30). In Southern Asia, the region Bangladesh belongs to, about 7.9% of women in the general population are estimated to harbor cervical HPV infection at a given time and 82.8% of invasive cervical cancers are attributed to HPV 16 or 18 (31).
Mutation that can induce neoplastic transformation is known as oncogenic mutation and can produce an abnormal protein or change expression level of the protein coded by the gene harboring the mutation. This abnormal or over expressed protein can be a potential target of drugs. This study attempted to identify mutations in genes which can be specific for cervical cancer.
In this study, we amplified specific gene fragments. EGFR, KRAS and PIK3CA genes were selected to be amplified in specific regions since these genes are implicated in most cancers, especially in cervical cancers. Total 5 pairs of primers were used. Two different sets of primers were chosen to amplify two different sites on PIK3CA and KRAS genes since these two genes were to harbor more than one mutation hotspot.
We observed that, among 46 samples, 26 samples harbored mutation (56.52%). The other samples did not harbor any mutation. Among the 26 samples which harbored mutation, 22 samples harbored mutation in one gene (84.62%) whereas 4 samples harbored mutations in more than one gene (15.38%).
Eleven different mutations were found in amplified EGFR gene fragments, among which 1 specific mutation was found in more than one patient samples (in 7 samples). On the other hand, 24 different mutations were found in PIK3CA gene fragment amplicons, among which 2 different mutations were found in 6 patients. Four different mutations were found in KRAS gene fragment amplified products.
Almost all the mutations identified were base substitution. Both transitions and transversions were found. Only one insertion mutation was found in EGFR gene fragment at chromosomal position 55,170,230 where an adenine residue was found added.
Our study shows that Bangladeshi patients have KRAS mutation frequency (8.7%) similar to that reported by Wright et al. (16) (8.8%). A 7% mutation in KRAS was also reported by Spaans et al., 2015 (32). Iida et al., 2011 (33) also reported somatic mutations in KRAS in 3 (6.3%) of 48 cervical adeno/adenosquamous cell carcinomas.
However, surprisingly, EGFR mutation frequency is over 6 times higher in our patients (23.91% versus 3.8%) and PIK3CA mutation frequency is over 1.6 times (52.17% versus 31.3%) compared to study of Wright et al. (16), 2013 and 2.6 times higher (52.7% versus 20%) compared to Spaans et al., 2015 (32), report. Even PIK3CA mutation rate is 3.8 times higher than those in Chinese patients (13.6%) as reported by Xiang et al., 2015(34).
Survival of patients with KRAS mutation is poorer than in patients without KRAS mutations (35), therefore, a combination of KRAS mutation detection and HPV genotyping would be useful in identifying a patient with poor prognosis for further interventions. Among the three most common histological subtypes of cervical cancer (squamous cell carcinoma (SCC), adenocarcinoma (AC), and adenosquamous carcinoma (ASC)) KRAS mutations are reported to occur more frequently in AC than SCC (32). The same study also observed worse disease-free survival (HR 1.57, P=0.043) in positive KRAS mutation cases. A set of endometrial-like cervical cancers comprised predominantly of HPV-negative tumors and characterized by mutations in KRAS, ARID1A and PTEN was discovered in a study conducted by (36).
EGFR is a membrane tyrosine kinase receptor that is known to contribute to the growth activity and tumor survival, and hence this has become a therapeutic target in several cancers. In EGFR gene, exons 18–21 are the hot spot region for gain-of-function mutations. Previous studies (33, 37) found a strong correlation between poor prognosis and EGFR gene amplification in patients with cervical squamous cell carcinoma. In other carcinomas like leukemia, glioblastoma, and colorectal, gastric, breast, and hepatocellular carcinomas EGFR mutation frequency reported to be low. Neither Iida et al., 2011 (33); nor the study of Arias-Pulido et al., 2008 (37) found presence of EGFR mutations in exons 19 and 21. No mutations identified in their samples affecting the EGFR kinase domain in exons 18 through 21 in human neoplastic samples analyzed. However, in cervical carcinoma, we have found a mutation in our Bangladeshi patients. These suggest that mutations in the EGFR kinase domain may be not common in other part of the world. Our results suggest, therefore, that treatment of CC patients with TKIs needs mutational screening before prescribing drugs and may not have the same efficacy as seen in patients with no-mutation. Therefore, CC patients without such mutation, targeting the EGFR with other inhibitors may be more appropriate.
PIK3CA mutations can cause the deregulation of the PI3K/Akt signaling pathway, which comprises cell proliferation, transformation, and cell survival, stimulating oncogenesis. Aberrations in this pathway are described in various cancers, including cervical cancer, and this has led to the development of PI3K-inhibitors and Akt-inhibitors as potential cancer therapies, with some already having reached clinical trials. PIK3CA mutation rates are very heterogeneous in different studies (20–37%) (38, 39). However, Bangladeshi patients harbor more mutations (52.17%) in this gene. Spaans et al., 2015 (32) detected a clear trend for reduced survival in patients carrying a PIK3CA mutation, especially with the SCC subtype. Similarly Wright et al., 2013 (16) also showed that an association lies between PIK3CA mutation and shorter survival.
After DNA sequence analysis, corresponding amino acid analysis was done to find out the effect of mutations on proteins. Among the 17 different mutations, 8 mutations were located in exon regions. Six of them were PIK3CA mutations and the other 2 were EGFR gene mutations. The other 9 mutations were located in introns; hence, they did not have any effect on the overall amino acid sequence compositions.
These eight exonic mutations were analyzed and it was found out that 6 of them were synonymous. Among the rest 2 non-synonymous mutations, one mutation (A>G) in EGFR gene located at 55,170,332 in chromosome 7 results in a methionine to valine substitution at amino acid position 636 of the protein; while the result of the non-synonymous mutation (T>A) in PIK3CA gene located at 179,218,237 in Chromosome 3 is a substitution of leucine to isoleucine at amino acid position 523 of the protein.
We have done different bioinformatic analysis of mutations we have found using tools like Variant Effect Predictor tool (20), MuPRo tool (24), MutationMapper tool (25) etc. LoFtool (21) scores of all intronic and exonic mutations which were significantly low, predicts that effect of the mutation on the functionality of the gene is probably damaging. SIFT (22) and polyphen (23) scores showed that effect of the non-synonymous mutation on the function of protein is not that much significant. But the stability of protein may be affected which is predicted by MuPRo tool (24). Further in vitro protein level analysis needs to be carried out to find out the exact effect of this amino acid change.
We were particularly interested in the few selected mutation hotspots only, as these regions tend to accumulate a high frequency of mutations compared to the other regions of the target genes. It is also true that other significant mutations can also be found in the other regions of the target genes. But in this study, we only wanted to check the profiles of the mutations which were previously reported to establish the already reported mutations as a biomarker for cervical cancer diagnosis. However, taking cue from our results, the full length of the target genes can also be taken into considerations for future studies. Furthermore, we had a limitation of smaller sample size and because of that no statistically significant association between the mutations and clinicopathological features of the patients was observed.