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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 - BMC Med (2015)

Bottom Line: Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Molecular Biology, Department of Biological Sciences, Diadema Campus, Federal University of São Paulo, Rua Pedro de Toledo, 669, São Paulo, SP, 04039-032, Brazil. ca_oak@yahoo.com.br.

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.

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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.
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Fig5: 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.

Mentions: The Youden index obtained from ROC curves was used to determine the cutoff values for upregulation of miR-200a, miR-200c, miR-203, and miR-205, which varied from 1.54 to 5.96 (Table 2, Figure 4). The expression profile of these four microRNAs in the metastatic and non-metastatic FFPE lymph node samples showed they were highly associated with the presence of metastatic cells in the neck lymph nodes of HNSCC cases (miR-200a, 76%; miR-200c, 88%; miR-203, 100%; miR-205, 100%; Figure 5, Table 2). However, miR-200a and miR-200c barely detected the presence of micrometastases (40% and 80%, respectively), and failed in detecting cases with isolated tumor cells. On the other hand, miR-203 and miR-205 displayed high sensitivity levels (100%) and were also able to correctly classify lymph nodes containing macrometastases, as well as micrometastases or isolated tumor cells (Table 2).Table 2


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 - BMC Med (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493814&req=5

Fig5: 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.
Mentions: The Youden index obtained from ROC curves was used to determine the cutoff values for upregulation of miR-200a, miR-200c, miR-203, and miR-205, which varied from 1.54 to 5.96 (Table 2, Figure 4). The expression profile of these four microRNAs in the metastatic and non-metastatic FFPE lymph node samples showed they were highly associated with the presence of metastatic cells in the neck lymph nodes of HNSCC cases (miR-200a, 76%; miR-200c, 88%; miR-203, 100%; miR-205, 100%; Figure 5, Table 2). However, miR-200a and miR-200c barely detected the presence of micrometastases (40% and 80%, respectively), and failed in detecting cases with isolated tumor cells. On the other hand, miR-203 and miR-205 displayed high sensitivity levels (100%) and were also able to correctly classify lymph nodes containing macrometastases, as well as micrometastases or isolated tumor cells (Table 2).Table 2

Bottom Line: Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Molecular Biology, Department of Biological Sciences, Diadema Campus, Federal University of São Paulo, Rua Pedro de Toledo, 669, São Paulo, SP, 04039-032, Brazil. ca_oak@yahoo.com.br.

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.

Show MeSH
Related in: MedlinePlus