Diagnostic Value of Circulating microRNAs in Laryngeal Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis

Background : Circulating microRNAs (miRNAs) play an important role in the biological processes of cancers and are promising biomarkers of cancer diagnosis. Objectives : To estimate the diagnostic value of microRNAs in laryngeal squamous cell carcinoma (LSCC) patients, comparing with the non-LSCC controls. Population : Six eligible LSCC studies including 1,585 LSCC patients with corresponding 2,095 non-LSCC controls from years 2000 to 2017 were analyzed. Methods : Medline, Epub Ahead of Print, In-Process & Other Non-Indexed Citations, EMBASE, Cochrane Library and Web of Science were searched in this study. We conducted a meta-analysis to identify studies that reported the diagnostic data of miRNAs both in LSCC patients and controls. I n addition, we evaluated and compared the diagnostic value of upregulated miRNAs with downregulated miRNAs . Results : Six studies with corresponding specificity and sensitivity data were included in this study. The pooled sensitivity, specificity and AUC were 0.89 (95% CI: 0.79-0.94), 0.87 (95% CI: 0.77-0.93), and 0.94 (95% CI: 0.92-0.96), respectively. However, the heterogeneities of these studies were quite high, the value of I 2 for the pooled sensitivity, specificity were 96.82% and 97.08%, respectively. Subgroup analysis of upregulated and downregulated miRNAs showed a similar diagnostic value but the heterogeneity remained high. Publication bias was found in Funnel plot of pooled and upregulated miRNAs, while not obvious in downregulated miRNAs. Conclusions : Circulating miRNAs showed diagnostic significance in laryngeal cancer, however, the results of this meta-analysis revealed significant heterogeneity. Therefore, the diagnostic value of miRNAs in LSCC seems limited.

diagnosis of LSCC depends on pathological findings, imaging examination and laryngoscopies, however, no reliable biomarker has been identified to screen or diagnose LSCC [5].
MicroRNAs (miRNAs) are small non-coding, single stranded RNAs containing about 22 nucleotides. They regulate gene expression by influencing the 3'-UTP-binding of target genes at transcription or post-transcription level. Over the past decades, plenty of studies demonstrated that miRNAs played critical roles by controlling the expression of targeted mRNAs; studied the mechanism of miRNAs in affecting the occurrence and development of LSCC; and identified that miRNAs had effects on the biological processes, proliferation, invasion and metastasis in tumors [6].
Koichiro Saito et al. reported that miR-196a was cancer-specific expressed in LSCC [7]. MiR-331-3p, miR-603, miR-1303, miR-660-5p and miR-212-3p were reported to be only detected in the plasma of LSCC [8]. These previous studies demonstrated that circulating miRNAs were capable of becoming potential less-invasive LSCC diagnostic biomarkers, while the comprehensive diagnostic value of miRNAs for LSCC is still unclear.
This systematic review and meta-analysis included all qualified studies, evaluated the differential expression of candidate circulation miRNAs in LSCC and summarized their roles as biomarkers to diagnose LSCC.

Methods: Protocol and registration
This systematic review was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) checklist [9], with PROSPERO registration number of 159381. squamous cancer; (b) case-control or cohort study design; (c) pathology-proven laryngeal carcinoma; (d) providing the sensitivity and specificity of diagnostic value, ROC curve and AUC with 95% confidence interval (CI), and (e) providing detailed information about the recruitment of participants, diagnostic protocols, genotyping, statistical analysis and other relevant methodological data.
The exclusion criteria included: (1) non-English papers; (2) case reports, letters, historical reviews; (3) studies performing on animal samples; (4) studies without specific diagnostic data (5) studies without control group of non-LSCC population; (6) studies repeated or overlapping publications with the same author or team.

Data Extraction
Data extraction was performed independently by two investigators (DNC and WDP). In case of any disagreement, a consensus was reached by a consultation among all authors. The data we extracted from those included studies were: (1) basic characteristics, including the first author, publication year, country, ethnicity, male ratio, source of control, cancer type, miRNA profiling type, sample type, method of miRNAs detection; (2) diagnostic value, including (i) sensitivity and specificity; (ii) ROC curve; (iii) AUC with 95% CI.

Assess Bias Across Individual Studies:
The qualities of studies, especially the quality of diagnostic accuracy, were assessed independently by 2 reviewers using Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) criteria [10]. The QUADAS-2 tool contains 4 key domains: patient selection, index test, reference standard, and flow and timing, which gives a maximum score of 7 (Fig. 5).

Statistics Analysis
All statistical analyses were conducted by using STATA version 15. Diagnoses of LSCC were based on the accuracy of the identified miRNAs. The bivariate meta-analysis models were applied to calculate the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). The sensitivity and specificity of all included studies were used to plot a summary receiver operator characteristic (SROC) curve, and the area under the SROC curve (AUC) was calculated in the meta-analysis (Fig. 2C, Fig. 3C, Fig. 4C). Similarity among studies was evaluated by chi square and I 2 statistics to evaluate and substantial heterogeneity was defined if I 2 was above 50%.

Results: Study selection and characteristics
We obtained 1,497 potential articles in the initial screening, among which, 826 duplicate studies were excluded( Figure 1). Two independent reviewers (DNC and WDP) assessed the remaining 671 studies by screening titles and abstracts, then excluded 658 unrelated articles: 1341 studies were ceRNAs, lncRNAs or other non-microRNAs; 1,714 articles did not involve laryngeal subsite; and 839 studies were systematic reviews, case reports, experimental studies, or commentaries. Subsequently, of the 17 full-text articles evaluated by DNC and WDP, 11 studies were excluded: six of them reported disease management without diagnostic value; two of them reported overall head and neck cancer without certain information for LSCC; three of them reported diagnostic graphs without eligible diagnostic data. Ultimately, six articles with 17 miRNAs reported met the inclusion criteria and included in this meta-analysis.

Diagnostic Value Of Circulating miRNAs
The diagnostic value of the 17 miRNAs in peripheral blood circulation of LSCC patients was analyzed as follows (Table 1). Using bivariate meta-analysis models, the pooled sensitivity, specificity and AUC The pooled results manifested that circulating microRNAs had a high diagnostic accuracy.
Although the significant heterogeneity was not observed between the subgroups according to Fig. 3B and Fig. 4B, the meta-regression was performed with some potential predictor variables: ethnicity (Asian vs. others), sample size (≥ 200 vs. <200) and miRNA profiling (single-vs. multiple-miRNAs assay). The statistical differences existed in ethnicity and sample size, indicating partly origin of heterogeneity.

Risk Of Public Bias Within Studies
The publication bias of the included studies was checked by Deeks' funnel plot test, which indicate that public bias exists in pooled studies and unregulated subgroup, while no significant public bias was observed in downregulated study( Figure S1, Figure S2, Figure S3).

Discussion:
Although larynx is part of the head and neck, the biological characteristics of laryngeal cancer are much different from cancer of the oral cavity and pharynx [17]. HPV or EBV infection would indicate a suspected oropharyngeal or nasopharyngeal cancer origin. P53 and EGFR are normally expressed in HNSCC according to recent studies, however, the mechanism in laryngeal cancer remains occult [17], and no specific biomarkers was identified to be related to laryngeal cancer diagnosis or screening [20].
Current gold diagnostic standard for LSCC patients is invasive pathologic biopsies, accompanying with CT, MRI, or PET-CT [5], and studies focusing on the diagnostic value of non-invasive marker like miRNAs have increased in recent years [21]. It was reported that miRNAs dysregulations were associated with many diseases, such as cancers [23], cardiovascular conditions, even mental disease [22]. There were increasing studies reporting associations between miRNAs and head and neck cancer, especially laryngeal carcinoma. Different miRNAs have potential capacity in serving as new diagnostic, therapeutic and prognostic biomarkers.
In this meta-analysis, the specificity and sensitivity of miR-31, miR-33 and miR-let-7 were extraordinarily high (up to 100%), indicating a high diagnostic efficiency. Yang et al. [27] confirmed that overexpression of miR-31 suppressed LSCC cell growth, cell cycle and cell invasion, besides, they demonstrated that miR-31 was downregulated in LSCC tumors and correlated with advanced cancer stage. Although no explicit mechanism has been confirmed between miR-33 or miR-let-7 in LSCC, miR-33 was reported to mediate proto-oncogene PIM3 repression [28]; as a key regulator of tumor metastasis, miRNA let-7a led to the enrichment of its target gene HMGA2 in tongue squamous cell carcinoma (TSCC) tissues and cell lines [29]. Additionally, the expression levels of miRNA-21, miRNA-145, miRNA-218 [30,31] were related to the degree of differentiation, lymph node metastasis and TNM staging, which would affect the prognosis of LSCC. Therefore, dysregulation of miRNAs plays a role in various signaling pathways or cell cycles, affecting the occurrence and development of laryngeal cancer.
In this meta-analysis, high efficacy of diagnostic value of miRNAs was pooled while significant heterogeneity was observed (I 2 = 96.08% for sensitivity, and 96.32% for specificity, P < 0.001). The possible explanations for the heterogeneity were: first, the ethnicity (Asia vs. others) and sample size(n ≥ 200 vs. n < 200) might induce the heterogeneity according to the results of meta-regression; second, various miRNAs might be applicable to diverse tumors type or stages on account of the different mechanism of each miRNA; third, no exact cutoff value had been uniformed to determine the dysregulation of miRNAs in LSCC. Moreover, publication bias existed in the pooled and the upregulated studies, but was not obvious in the downregulated subgroup. This study has limitations.
On one hand, we did not perform a meta-analysis for any exact miRNA due to the limited number of original studies. On the other hand, since the main source of heterogeneity could not be found in subgroup analysis and meta-regression, the potential discriminative ability of miRNAs as circulating biomarkers for LSCC diagnosis needs to be verified in future.

Conclusions:
In this meta-analysis, we recognized that miRNAs have a certain diagnostic role in LSCC, however, the results of this meta-analysis revealed significant heterogeneity, indicating the diagnostic value of miRNA is currently limited.

Availability of data and material:
All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests:
The authors declare that they have no competing interests.

Authors' contributions:
JJR and DNC designed the study; DNC and WDP analyzed and interpreted data in including studies; DD and KQ performed quality control of data and algorithms; WJY, YFR and XHY performed the statistical analysis; YS and WY were involved in the preparation of the manuscript; DNC and WDP were major contributor in writing the manuscript; JJR and YZ reviewed the manuscript. All authors read and approved the final manuscript       Investigators' quality assessment for included studies using the QUADAS-2 assessment. (A) Graph; (B) Summary.

Supplementary Files
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