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BMC Med
2015 May 09;13:108. doi: 10.1186/s12916-015-0350-3.
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Accuracy of microRNAs as markers for the detection of neck lymph node metastases in patients with head and neck squamous cell carcinoma.
de Carvalho AC
,
Scapulatempo-Neto C
,
Maia DC
,
Evangelista AF
,
Morini MA
,
Carvalho AL
,
Vettore AL
.
Abstract
BACKGROUND: The presence of metastatic disease in cervical lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients is a very important determinant in therapy choice and prognosis, with great impact in overall survival. Frequently, routine lymph node staging cannot detect occult metastases and the post-surgical histologic evaluation of resected lymph nodes is not sensitive in detecting small metastatic deposits. Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers. Herein, we evaluated the feasibility of using the expression of microRNAs to detect metastatic cells in formalin-fixed paraffin-embedded (FFPE) lymph nodes and in fine-needle aspiration (FNA) biopsies of HNSCC patients.
METHODS: An initial screening compared the expression of 667 microRNAs in a discovery set comprised by metastatic and non-metastatic lymph nodes from HNSCC patients. The most differentially expressed microRNAs were validated by qRT-PCR in two independent cohorts: i) 48 FFPE lymph node samples, and ii) 113 FNA lymph node biopsies. The accuracy of the markers in identifying metastatic samples was assessed through the analysis of sensitivity, specificity, accuracy, negative predictive value, positive predictive value, and area under the curve values.
RESULTS: Seven microRNAs highly expressed in metastatic lymph nodes from the discovery set were validated in FFPE lymph node samples. MiR-203 and miR-205 identified all metastatic samples, regardless of the size of the metastatic deposit. Additionally, these markers also showed high accuracy when FNA samples were examined.
CONCLUSIONS: The high accuracy of miR-203 and miR-205 warrant these microRNAs as diagnostic markers of neck metastases in HNSCC. These can be evaluated in entire lymph nodes and in FNA biopsies collected at different time-points such as pre-treatment samples, intraoperative sentinel node biopsy, and during patient follow-up. These markers can be useful in a clinical setting in the management of HNSCC patients from initial disease staging and therapy planning to patient surveillance.
Figure 1. Schematic representation of lymph nodes sectioning protocol for immunohistochemistry (solid line) and molecular (dotted line) analyses. (A) Non-metastatic lymph nodes: all sections at different levels were used for RNA extraction and subsequent molecular analysis (arrows). (B) Metastatic lymph nodes: only sections of the positive level at IHC analysis were used for RNA extraction and subsequent molecular analysis (arrow). The leftover material (regions marked with “*”) removed from each interval of 50 μm was collected and used in the analysis of the leftover material.
Figure 2. Immunohistochemistry staining for cytokeratins (M3515, clone AE1/AE3, Dako) in histologically negative lymph nodes of HNSCC patients. (A) Lymph node without evidence of metastases. (B) Lymph node with macrometastases. (C) Lymph node with micrometastases. (D) Lymph node with isolated tumor cells. A, B, and C: 40× magnification; D: 400× magnification.
Figure 4. ROC (Receiver Operating Characteristic) curves obtained for microRNAs miR-200a, miR-200c, miR-203, and miR-205 in the FFPE validation set.
Figure 5. Expression profile of miR-200a, miR-200c, miR-203, and miR-205 in lymph node samples containing macrometastases (Ma; n = 18), micrometastases/isolated tumor cells (Mi and isolated tumor cells; n = 7), or in non-metastatic lymph node specimens (NM; n = 13). The Y-axis shows the log10 fold-change of the relative expression (2-ΔΔCt). The P value (Mann–Whitney) from each comparison is provided. The dotted line indicates the cutoff adopted according to the Youden index (value in which the difference between sensitivity and 1-specificity is maximum) obtained from the ROC curves analysis. The horizontal line indicated the median of fold-change values for each group.
Figure 6. Expression profile of miR-203 and miR-205 in FNA biopsies from lymph nodes classified as positive (FNA+) or negative (FNA–) according to (A) cytological diagnostic (FNA+: n = 42; FNA–; n = 71) and (B) histological diagnostic (FNA+: n = 45; FNA–; n = 68). The Y-axis shows the log10 fold-change of the relative expression (2-ΔΔCt). The P value (Mann–Whitney) from each comparison is provided. The dotted line indicates the 10-fold cutoff adopted.
Figure 7. Receiver Operating Characteristic curves obtained for microRNAs miR-203 and miR-205 in in FNA biopsies from lymph nodes classified as positive (FNA+) or negative (FNA–) according to (A) cytological diagnostic and (B) histological diagnostic.
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