Serum Exosome MicroRNA As Novel Biomarkers For The Prediction Of Recurrence In Patients With Ⅱ/Ⅲ Colon Cancer

Colorectal cancer is the third most common cancer and the third leading cause of cancer death in the USA.One half of all colon cancer patients will present with stage Ⅱ or stage Ⅲ disease;moreover,20%-50%of them will develop disease recurrence,74%being within the first 3 years of diagnosis despite surgical intervention and adjuvant chemotherapy.Although,clinicopathological risk factors such as T4 and/or N2 disease,poorly differentiated histology,or suboptimal lymph node dissection may be considered a poor prognostic factor for recurrence,predictive markers for patients likely to benefit from adjuvant chemotherapy have not been clearly identified.As a result,the recommended role of adjuvant chemotherapy has remained unchanged for the past decade for all stage Ⅲ colon cancer patients.Yet,the role of adjuvant chemotherapy in stage Ⅱ colon cancer is poorly delineated.The identification of biomarkers which could add prognostic and predictive value to existing clinicopathological risk factors are urgently needed for locally advanced disease,notably stage Ⅱ colon cancer.Despite surgical intervention,20%-50%of stage Ⅱ and stage Ⅲ patients will develop recurrent disease,74%being within the first 3 years of diagnosis.48,3 For stage Ⅱ patients,current treatment guidelines do not advocate the use of adjuvant chemotherapy.For stage Ⅱ patients,high-risk features for disease recurrence include colon obstruction,T4 disease,<10 lymph nodes surgically dissected,Iymphvascular invasion,and poorly differentiated histology.Adjuvant oxaliplatin-based therapy may be beneficial in stage Ⅱ patients with these high risk features but is currently not the standard of care.10 Recently,QUASAR collaborative group has advocated the benefit of chemotherapy in all stage Ⅱ patients following surgical resection although the optimum regime has not been identified.51 Currently,adjuvant oxaliplatin-based chemotherapy is approved for all stage Ⅲ colon cancer patients and has been determined to result in a 24%decreased risk of recurrence.Despite the use of adjuvant chemotherapy only a 10%absolute improvement in overall survival has been established.49 Given this marginal benefit in overall survival,the role of adjuvant therapy remains controversial.It is presumed the etiology for recurrent disease is attributed to microscopic disease that is not self-evident upon surgical or radiological assessment.Therefore,adjuvant 5-FU based chemotherapy is provided to prevent any manifestations of recurrent tumor or metastatic disease.Existing heterogeneity in stage Ⅱ and Ⅲpatients attributed to surgical technique,the number of lymph nodes involved and removed,colonic obstruction or visceral perforation,histology,age at presentation,coexisting morbidities,and molecular foundation of the tumor confound the medical oncologist’s perspective of what is presumed to be a simple categorical AJCC stage Ⅱor stage Ⅲ patient.AJCC stage Ⅲ is further subdivided into subgroups of A,B,or C based primarily on the T and N stage but not based on a summation of potentially preexisting poor prognostic features.Intuitively there are patients who are inherently resistant to 5-FU or oxaliplatin-based chemotherapy and may derive little benefit from 6 months of adjuvant treatment.Furthermore,although incorporating oxaliplatin is currently commonplace in our adjuvant chemotherapy regimen this may also come at the cost of increased treatment-related toxicities.MicroRNAs(miRNAs),the small noncoding RNAs that are associated with the development of cancer,have been shown to be potential biomarkers in various types of cancer including colon cancer.Examination of miRNA profiles in tumor versus normal tissues or in circulating blood have discovered various miRNAs in association with tumor progression or patient survival.Exosomes have been attracting major interest as potential diagnostic and prognostic biomarkers of cancer.Exosomes are secreted lipid-bilayer-enclosed vesicles of 50-150 nm diameter which can be secreted by multiple cell types,including cancer cells,and can be isolated from many body fluids,including both plasma and serum.Exosomes may contain DNA,RNA and protein molecules and maintain the integrity of these contents in circulation.Tumor-derived exosomes are emerging as mediators of metastasis.Isolation of cancer specific exosomes in body fluids could enable the identification of DNA,RNA,miRNA,and proteins,and aid in the treatment and management of cancer.The role of serum exosomal miRNA as a prognostic marker for colon cancer recurrence has been explored in one reported study,which was conducted in six patients with or without tumor recurrence using microarray technology.One miRNA,i.e.miR-19a was identified as a prognostic marker.To systematically evaluate the prognostic or predictive value of serum exosomal miRNA in patients with stage Ⅱ or Ⅲ colon cancer,we conducted RNA-sequencing scanning on serum exosomal miRNAs followed by a quantitative reverse-transcription PCR(qRT-PCR)assay using baseline blood samples of a cohort of 84 stage Ⅱ and stage Ⅲ colon cancer patients.The expression profiles of mature miRNA in patients with recurrent disease were compared with those in patients without tumor recurrence.The etiology of colon cancer is most commonly sporadic,but may also be hereditary as in familial adenomatous polyposis(FAP)and hereditary non-polyposis colorectal cancer(HNPCC).Regardless,the development of colon cancer involves genetic mutations leading to the progression of normal epithelial cells of the intestinal mucosa from adenoma to carcinoma.Due to this well-established sequential transformation,there are multiple opportunities to interfere with the natural course of the disease.This may take the form of screening,chemoprevention,ehemotherapy,surgical resection,or palliative therapy.Prognosis depends on the cancer stage at the time of diagnosis.The Tumor Node Metastases(TNM)staging system of the American Joint Committee on Cancer/Union for International Cancer Control is the preferred staging system for colon cancer.Detection of early stage colon cancer may confer a 90%5-year survival rate,compared to 12%if distant metastasis has occurred.Given that symptoms are often not obvious,detection of colon cancer relies heavily on screening.Over the past two decades,the introduction of screening programs such as endoscopy,fecal occult blood testing(FOBT),and barium enema have led to improved early detection of colon cancer,which has shown a reduction in colon cancer incidence and mortality in many countries.Unfortunately,less than 40%of colon cancer patients are identified early enough in the disease when management is most efficacious.In addition,adherence to such screening programs is insufficient at nearly 50%in high-risk patients.Endoscopy is invasive and expensive,whereas the less invasive and less expensive FOBT had a sensitivity of only 47%-73%in case control studies.Thus,there is a strong necessity for the development of accurate and non-invasive markers for the diagnosis and prognosisof colon cancer.Recent studies have implicated microRNA(miRNA)as serving a key part in the colon cancer progression and outcome.This article focuses on the utility of miRNA inthe diagnosis and prognosis of colon cancer,as well as its role inpredicting sensitivity to chemotherapy.We hypothesize that patients with recurrent disease will possess germlite polymorphisms involved in 5-FU and oxaliplatin metabolism.However,this alone may not account for all instances of recurrent disease.The use of tissue proteomics validated by serum proteomics may provide an adjunctive means to identify this patient population.We believe that a comprehensive analysis of genomics,proteomics,as well as environmental factors,will allow us to discover potential prognostic indicators other than CEA.The long-term goal of our research project is to develop novel strategies to identify biomarkers that may determine patients who are at increased risk of recurrence culminating in the creation of a screening and/or prevention study in stageⅡ and/or Ⅲ colon cancer patients.To reach this goal,we will take a multi-step approach.First,we will try to identify the genetic,proteomic,and environmental factors that impact tumor recurrence.The data from this study may serve as the foundation for the creation of a colon cancer predictive model in a large-scale cooperative group study at a later date.We hope these results will change the paradigm of evaluating and treating all stage Ⅱ/Ⅲ colon cancer patients in a homogenous manner and apply it individually to each patient.Serum exosomal miR-4772-3p as a predictor for tumor recurrence in stage Ⅱ and Ⅲ colon cancerPurpose:The aim of this study was to evaluate serum exosomal miRNAs as prognostic biomarkers for tumor recurrence in stage Ⅱ or Ⅲ colon cancer.Methods:Blood samples were collected from 84 patients with stage Ⅱ or Ⅲ colon cancer after tumor resection and before consideration of adjuvant therapy.Serum exosomal RNA was extracted and miRNA profiles were determined by RNA-sequencing in pooled RNA samples from 20 patients each in the recurrent and non-recurrent group.Differentially expressed mature miRNAs between the two groups were identified and the top hits were validated in individual RNA samples from 27 patients with recurrent and 57 without recurrent disease using quantitative real-time PCR(qRT-PCR).Results:A total of 145 differentially expressed mature miRNAs with nominal significance(P<0.05)and 50 with false positive discovery rate(FDR)<0.05 were identified.A validation study by qRT-PCR of ten top hits showed that miR-4772-3p was significantly under-expressed and miR-4732-5p was over-expressed in patients with recurrent disease compared to those without recurrent disease,P=0.002 and 0.044,respectively.Logistic regression and ROC analysis demonstrated better predicting power of mir-4772-3p than clinical predictors.Patients with a lower level of miR-4772-3p expression had a 3.38-fold(95%Cl:1.19-9.61)higher risk for tumor recurrence(P=0.022).ROC analysis showed AUC of 0.72(95%Cl,0.59-0.85),P=0.001 and sensitivity and specificity of 78.6%and 77.1%,respectively.Conclusions:Reduced expression of miR-4772-3p in serum exosomes was identified as a potential prognostic biomarker for tumor recurrence in stage Ⅱ or stage Ⅲ colon cancer patients.