| BackgroundIn the past decade, knowledge about the biology of cancers has increased greatly, and there have been considerable improvements of cancer biomarkers. Ideally, biomarkers should be easily accessible such that they can be sampled non-invasively. The goal of the cancer biomarker field is to develop simple non-invasive tests that indicate cancer risk, allow early cancer detection, classify tumours so that the patient can receive the most appropriate therapy and monitor disease progression, regression and recurrence. The dynamic nature of the circulatory system and its constituents reflects diverse physiological or pathological states, and the ease with which the blood can be sampled makes it a logical choice for biomarker applications.Previous studies found that the changes of gene or protein cancer biomarkers lack specificity, and the changes mainly occurred in tumor cells. Although the use of these biomarkers significantly increase the accuracy of diagnosis, they are limited in the clinical setting. Further challenges include the complexity of protein composition, posttranslational protein modifications, proteolysis and protein denaturation as well as the low abundance of proteins of interest or the difficulty of developing suitable high-affinity capture reagents.MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs of 18～25 nucleotides in length that modulate gene expression post-transcriptionally by targeting the 3’untranslated region (3’UTR) of its target mRNA. Recent evidence has shown that about half of the human miRNAs are located in cancer-associated genomic regions, including minimal regions of LOH, minimal regions of amplification and fragile sites. Their abnormal expression plays a key role in the hallmarks of cancer, such as cell proliferation, differentiation, apoptosis, migration, invasion, metastasis and angiogenesis. These miRNAs have been confirmed to downregulate the expression of many cancer-related genes and function as oncogenes and tumor suppressor genes.miRNAs might be an especially rich source of diagnostic, prognostic and predictive information as biomarkers in cancer because of their fundamental role in development and differentiation and their pervasive corruption in the lineage-dependent mechanisms that underlie tumorigenesis. Multiple studies have already explored the potential value of tumor-specific miRNAs profile as cancer biomarkers in diagnosis, prognosis, and response to treatments. Compared with tissue miRNA profiling, the promising potential of plasma miRNAs as cancer biomarkers is much better. In 2008, Lawrie CH et al for the first time reported that the plasma level of miRNAs was desregulated in cancer patients compared with normal control.Multiple studies have found that under normal healthy conditions, miRNAs circulate in the peripheral blood and can stimulate cellular signaling to contribute to biological homeostasis. In healthy donors, the circulating miRNAs are mainly serected from mononuclear cells and platelet, and the majority of miRNAs expressed in the blood are predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs are also predicted to be important modulators of immune function. It is well-known that the presence of miRNAs derives from the exosomes of cell lines. Exosomes containing miRNAs enable intercellular and interorgan communication in the body, suggesting a new mechanism of cell-cell communication. In fact, cancer cells also could release exosomes containing miRNAs into the peripheral blood, which lead to the desregulated expression of plasma miRNAs profiling. These deregulated miRNAs have been shown to function in signaling events that contribute for the development of tolerance, tumorigenesis and organ-specific metastasis. Review of the current literature suggests that plasma miRNAs could emerge as revolutionary source of blood-based biomarkers for cancer detection for several reasons:(1) expression of miRNAs is frequently dysregulated in cancer and it seem to be tissue-specific, (2) miRNAs show an unexpectedly high stability in serum and plasma, and (3) the non-invasiveness and simplicity of obtaining serum samples, (4) deregulation of miRNA is happened earlier than genes or proteins and more easily be founded. Thus, plasma miRNAs have demonstrated great potential as powerful and non-invasive cancer biomarkers.Recently, the studies of plasma miRNA profiling mainly focus on colorectal, breast, prostate cancer, non-small-cell lung cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, head and neck cancer. Multiple studies have already explored the potential value of plasma miRNAs as cancer biomarkers for cancer diagnosis, prognosis, and response to treatments and opened up the exciting prospective of using plasma miRNAs as powerful and non-invasive cancer biomarkers.Nasopharyngeal carcinoma (NPC) is one of the most common cancers in South China. NPC is characterized by its confined geographical prevalence, close association with Epstein-Barr virus (EBV) infection, and strong tendency of metastasis. Because the clinical presenting features of NPC are often nonspecific, and examination of nasopharynx requires expertise and renders early detection very difficult. EBV serology, including IgA antibodies against viral capsid antigen (VCA-IgA), early antigen (EA-IgA) and Plasma EBV DNA, have been found to be useful markers in clinical practice and correlate well with disease status. However, the serological tests have limited roles in monitoring disease progress and detecting recurrent diseases. Until now, there has been no efficient biomarkers confirmed by FDA for NPC in the clinical setting. Therefore, identifying more sensitive, unique and personalized biomarkers for cancer diagnosis and prognosis prediction remains urgent and significant.Although miRNA signatures for NPC have been established, elucidation of the role of deregulated miRNAs in NPC carcinogenesis remains in the early stage of development. More importantly, the majority of these reports are basic research, and only several reports with small sample sizes have clinical significance. However, there has no report about plasma miRNA profiling of NPC yet. In this study, we will examine the expression of several miRNAs in the plasma samples of NPC patients collected from 2007. Based on the detailed clinical data of these samples, we will investigate the clinical value of these deregulated miRNAs in NPC development.Materials and Methods1. Clinical dataFrom December 2007 to June 2011, the 217 blood samples of NPC patients were collected from Department of Otolaryngology-Head and Neck Surgery of Nanfang Hospital. Pathologists confirmed the histopathological diagnosis for each specimen. The histopathological types of NPC included nonkeratinizing undifferentiated carcinoma and keratinizing undifferentiated carcinoma. Tumors were staged according to the Chinese 2008 staging system for NPC. No patient received chemotherapy or radiotherapy before blood sampling. The quantification of plasma EBV-DNA was performed in the Clinical Laboratory of Nanfang Hospital. The treatment was according to the Guidelines in Oncology-v.2.2008 NCCN. The effect of induction chemotherapy was evaluated by the extent of diminution of metastatic lymph node. The side effects of chemotherapy was determined by Grading standard of acute and sub-acute toxicity reaction of anticancer drugs of WHO, and the digestive side effect was classified into 0-4 grades. The blood routine was evaluated by the difference of WBC number before and after chemotherapy. The follow-up of all patients were record, and the therapeutic effect was determined by a video laryngoscope examination and an iconographic examination. A total of 41 blood samples of the re-examination patients were collected.2. Plasma CollectionWhole blood (6-8 ml) was drawn into EDTA-containing tubes and separated into plasma and cellular fractions.73 control included the patients of chronic suppurative otitis media and chronic sinusitis. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital and registered in ClinicalTrials.gov (No. NCT01171235).3. Samples processingBlood samples were centrifuged to spin down the blood cells, and the supernatant was transferred into microcentrifuge tubes, followed by second centrifugation completely remove cellular components. Plasma was then aliquoted and stored at -80℃ until use. Blood samples were processed and plasma was frozen within 4 h of the blood draw.4. qRT-PCRTotal RNA was polyadenylated and reverse transcribed to cDNA. Real-time PCR was performed in duplicate measurements using SYBR Green PCR kit. The expression levels of 21 miRNAs were normalized to U6 snRNA, and were calculated utilizing the 2-ΔΔCt method. Cel-miR-39 was used as the positive control, and H2O was used as the negative control.5. Statistical analysisNonparametic test was used to determine the differences between plasma EBV-DNA and gender, digestive side effects and TNM and clinical classification, and the comparison between digestive side effects and the chemotherapy regimen was also determined by nonparametic test. One-way ANOVA was used for comparisons of the extent of diminution of metastatic lymph node and the decrease of WBC number, with the LSD tests for multiple comparisons. The comparison of ages between two groups was determined by Student’s t test. The difference of gender was determined by Chi-square test. Two-tailed Student’s t test was used for comparison of 2 independent groups (EBV-DNA positive and EBV-DNA negative groups). The relationship between the expression of 5 miRNAs and age, the extent of diminution of metastatic lymph node, the change of WBC number was determined by Pearson correlation. The expression of 5 miRNAs and TNM classification were compared using nonparametic test. The relationship between the expression of 5 miRNAs and the TNM and clinical classification was determined by Spearman rank correlation, which was also used to determine the correlation between plasma EBV-DNA and the digestive side effects of chemotherapy. Survival analysis was analyzed using a Cox regression model. Multivariate logistic regression model was established. Receiver-operating characteristics (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating between tumors and controls. The expression levels of 5 miRNAs before and after treatment was compared with paired t test. All statistical analysis was performed with SPSS 13.0 software. A p value of less than 0.05 was considered statistically significant.Results1. Clinical data(1) There were 149 (68.7%) male patients and 68 (31.4%) female patients. The oldest patient was 77 years, and the youngest was 17 years. The median age was 45.93±11.78 years.(2) According to the Chinese 2008 staging system for NPC, stage 1 included 10 cases, stage 2 included 52 cases, stage 3 included 69 cases and stage 4 included 86 cases (17 cases with distant metastasis).(3) The positivity of plasma EBV-DNA was 61.7%, and the positivity was much higher in male patients compared with female patients. Moreover, the level of plasma EBV-DNA was positively correlated with the TNM and clinical classification.(4) There were significant differences of the the extent of diminution of metastatic lymph node among the PF regimen, the TP regimen and the endostar combined with the PF regimen. Moreover, there was significant differences of the the extent of diminution of metastatic lymph node between the TP regimen and the nimotuzumab combined with the TPF regimen. Although the therapeutic effect of TPF regimen was better than TP and PF regimen, the difference was not significant (P=0.063 and 0.292) because of the small sample size. There was no significant difference between the side effects of different chemotherapy regimens, but the digestive side effect of female patients was much serious than that of male patients.2. The deregulated expression levels of miRNA(1) The plasma levels of 21 miRNAs were screened, and the results found that the levels of miR-16,21,24,155 and 378 were significantly deregulated.(2) The expression level of miR-21 was negatively correlated with the T staging, which suggested that miR-21 had considerable clinical value in diagnosing early-stage NPC. Morevoer, the expression levels of miR-16,21 and 24 were negatively correlated with the N staging, which suggested this miRNA panel might be applicable as a marker to indicate the early metastasis of NPC.(3) The expression levels of miR-16,21,24, and 155 were negatively correlated with the plasma EBV-DNA, suggesting that these miRNAs might regulate each other with EBV.(4) There was no significant correlation between the expression of these five dysregualted miRNAs and the therapeutic effects as well as the side effects of chemotherapy.(5) The survival analysis of cox regression model showed that the expression of these five dysregualted miRNAs could not be used to predict the prognosis of NPC patients. Because the follow-up period of most patients was less than 3 years, the results should be further confirmed.(6) Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. The result showed that miR-21 yielded a AUC of 79.2%, and miR-155 yielded a AUC of 76.3%(sensitivity=75%, specificity=70%). The AUC for the 5 or 4 miRNA panel (miR-16,21,24,155 and 378) was 0.909 (sensitivity=87.7% and specificity=80.2%).3. The follow-up(1) The group were followed by 217 cases, with follow-up rate of 82% and up for 1 to 40 months.41 paired blood samples of before and after treatment were obtained. There are five patients with relapse or metastasis.(2) The expression levels of miR-16,21,24 and 155 were reduced after treatment in the patients without relapse and metastasis (P<0.05).(3) The expression levels of miR-21,24 and 155 were upregulated in the patients with relapse and metastasis. However, the expression of miR-378 significantly increased in the patients without relapse and metastasis, but its expression reduced in the patients with relapse and metastasis. The changing pattern of plasma miRNAs reflected the disease tendency.Conclusions1. Nasopharyngeal carcinoma is difficult to detect in early stage, and the blood EBV-DNA positivity of NPC patients remains low. However, the plasma EBV-DNA level is positively correlated with the TNM and clinical classification.2. The new class of anti-angiogenesis drugs and molecularly targeted drugs have better therapeutic effects with little side effects.3. The plasma levels of miR-16,21,24,155 and 378 are dysregulated in NPC patients compared with control.4. Plasma miR-21 has considerable clinical value in diagnosing early-stage NPC, and miR-16,21 and 24 may be applicable as a marker to indicate the metastasis of NPC.5. ROC analysis shows that only one miRNA (miR-16,21,24,155 and 378) provides a high diagnostic accuracy of NPC. Additionally, the more satisfactory diagnostic performance of the miRNA panel (miR-16,21,24,155 and 378) persists, and a higher diagnostic accuracy also could be achieved if miR-16 is excluded from the panel.6. The miRNA panel (miR-21,24,155 and 378) has the potential value as cancer biomarkers in prognosis and response to treatments. |
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