To study the role of pre‐treatment microRNA (micro ribonucleic acid) expression as a predictor of response to chemoradiation in locally advanced carcinoma cervix

Abstract Background Concurrent chemoradiotherapy followed by brachytherapy is the standard of care in locally advanced carcinoma cervix. There is no prognostic factor at present to predict the outcome of disease in locally advanced carcinoma cervix. Aim Differential expression of microRNAs can be used as biomarkers to predict clinical response in locally advanced carcinoma cervix patients. Methods Thirty‐two patients of locally advanced carcinoma cervix with International Federation of Gynecology and Obstetrics Stage IB‐IVA were enrolled from 2017 to 2018. Expression of microRNA‐9 5p, ‐31 3p, ‐100 5p, ‐125a 5p, ‐125b‐5p, and –200a 5p in formalin‐fixed paraffin embedded (FFPE) biopsied tissue were analyzed by real time quantitative reverse transcriptase polymerase chain reaction (RT qPCR). Pretreatment evaluation was done with clinical examination and MRI pelvis. All patients received concurrent chemoradiotherapy followed by brachytherapy. Patients were evaluated for the clinical response after 3 months of treatment, with clinical examination and MRI pelvis scan using RECIST 1.1 criteria. Patients with no residual disease were classified as Complete responders (CR) and with residual or progressive disease were classified as Nonresponders (NR). Results were statistically analyzed using Mann Whiney U test to examine significant difference between the expression of microRNA between complete responders (CR) and nonresponders (NR). Results microRNA‐100 5p was upregulated in complete responders (CR) which showed a trend towards statistical significance (p value = 0.05). Conclusion microRNA‐100 5p can serve as a potential molecular biomarker in predicting clinical response to chemoradiation in locally advanced Carcinoma cervix. Its role should be further investigated in a larger study population.


| INTRODUCTION
Cervical cancer is the fourth most common cancer worldwide with an estimated incidence of 570 000 cases in 2018. 1 With 31 1000 death in 2018, it is the fourth leading cause of cancer death worldwide. It is the most commonly diagnosed cancer in Sub-Sharan Africa and Southeastern Asia, where most patients present as locally advanced disease. External beam radiotherapy with concurrent chemotherapy followed by brachytherapy is the standard of care for locally advanced carcinoma cervix. 2 There is a higher risk of disease recurrence in patients presenting with locally advanced carcinoma cervix. Presently, there is no molecular marker in carcinoma cervix to predict the disease outcome.
MicroRNAs are a family of small noncoding RNA (Ribonucleic Acid) which control translation of messenger RNA (mRNA) into protein by posttranscriptional gene silencing. [3][4][5] Studies have shown that microRNA regulates thousands of human protein-coding genes. 6,7 MicroRNAs regulate many important biological processes such as cell division, cell differentiation, apoptosis, and cancer development. [8][9][10][11] Many groups have investigated the role of microRNA in cervical cancer, including Wang et al, who reported that microRNA-143 and -145 suppress cell growth and microRNA-146 promotes cell proliferation in cervical cancer. 12 Le et al identified that microRNA-29 acts as a tumor suppresser by inhibiting cell cycle progression and inducing apoptosis through YY1 and CDK6 protein. 13  -125a-5p, -100-5p, -125b-5p, and -200a-5p and -342 were expressed differentially in patients who respond to standard therapy of concurrent chemoradiotherapy than who do not respond to it. 16 In this study, we examined the expression profile of six micro-RNAs (microRNA-9 5p, -31 3p, -100 5p, -125a 5p, -125b 5p, and -200a 5p) in locally advanced carcinoma cervix. Differential expression of these six microRNAs was compared between patients who had a complete clinical response to standard treatment of chemoradiation to patients who did not respond to it.
With reference to previous studies, (p) = 50%, margin of error (d) = 8% of p and Z (1− alpha) = 1.96, a sample size of 38 cases were needed. In accordance with the journal's guidelines, we will provide our data for the reproducibility of this study in other centers if such is requested.

| RESULTS
During the study period six out of 38 patients were lost to follow up. So, we present here the analysis of results for 32 patients only.
The clinic pathological characteristics of the patients in the study are described in Table 1    breast cancer. 28 Similarly, microRNA-9 enhances the sensitivity of cells to ionizing radiation by suppression of NFκB1. 29 So, there is a probability that microRNA-200a and microRNA-9 will be upregulated in patients who respond to chemo-radiotherapy. In our study, we found microRNA- We propose that the results of our study should be validated in a study population with a larger sample size so that they could be used as predictors of response to chemoradiotherapy in locally advanced carcinoma cervix.

ACKNOWLEDGMENTS
No external funding was received for this study. We are very thankful to our patients who participated in the study.

CONFLICT OF INTERESTS
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

AUTHOR CONTRIBUTIONS
All authors contributed significantly to manuscript and inagreement with content of manuscript.

ETHICAL STATEMENT
The study design was approved by the institutional ethics review board before starting of study. Informed written consent was taken from every patient before recruitingAQ5 in the study. We have not reproduced any material from any other source.
This study and article submitted to Cancer Reports has been done in accordance to the guidelines of the journal and that is has been performed in an ethical and responsible way, with no research misconduct, which includes, but is not limited to data fabrication and falsification, plagiarism, image manipulation, unethical research, biased reporting, authorship abuse, redundant or duplicate publication, and undeclared conflicts of interest.

CONFLICT OF INTEREST
Authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.