Limits...
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.

Show MeSH

Related in: MedlinePlus

Heatmap representations of the 47 differentially expressed microRNAs with fold-change ≥2 and P value <0.05 (Rank Products) in the comparison between four metastatic lymph nodes (M: 7A, 1A, 8A, and 9A) and two non-metastatic lymph nodes (NM: 4C and 5C) resected from patients with T2N0 tongue squamous cell carcinomas. Non-supervised hierarchical clustering plotted based on the average ΔCt values. Up-regulated and down-regulated microRNAs are shown as red and blue, respectively. The columns represent samples and the microRNAs differentially expressed are shown in the lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Heatmap representations of the 47 differentially expressed microRNAs with fold-change ≥2 and P value <0.05 (Rank Products) in the comparison between four metastatic lymph nodes (M: 7A, 1A, 8A, and 9A) and two non-metastatic lymph nodes (NM: 4C and 5C) resected from patients with T2N0 tongue squamous cell carcinomas. Non-supervised hierarchical clustering plotted based on the average ΔCt values. Up-regulated and down-regulated microRNAs are shown as red and blue, respectively. The columns represent samples and the microRNAs differentially expressed are shown in the lines.

Mentions: Comprehensive microRNA profiles were generated for metastatic (n = 4) and non-metastatic (n = 2) lymph nodes from HNSCC patients using a quantitative RT-PCR array platform. From a total of 667 microRNAs, 439 were detected in at least two samples, regardless of the group, thereby serving as the pool of data for further analyses. From those, 61 presented a P <0.05 in the Rank Products test with 47 showing at least two-fold upregulation in all four metastatic samples. A non-supervised hierarchical clustering analysis using the ΔCt values of these 47 microRNAs displayed two distinct clusters formed by metastatic and non-metastatic lymph nodes (Figure 3). Finally, seven microRNAs (miR-200a, miR-200c, miR-203, miR-205, miR-382, miR-628-5p, miR-758), presenting more than 100-fold increment in the expression level in metastatic lymph nodes, were selected for further analyses (Table 1).Figure 3


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)

Heatmap representations of the 47 differentially expressed microRNAs with fold-change ≥2 and P value <0.05 (Rank Products) in the comparison between four metastatic lymph nodes (M: 7A, 1A, 8A, and 9A) and two non-metastatic lymph nodes (NM: 4C and 5C) resected from patients with T2N0 tongue squamous cell carcinomas. Non-supervised hierarchical clustering plotted based on the average ΔCt values. Up-regulated and down-regulated microRNAs are shown as red and blue, respectively. The columns represent samples and the microRNAs differentially expressed are shown in the lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Heatmap representations of the 47 differentially expressed microRNAs with fold-change ≥2 and P value <0.05 (Rank Products) in the comparison between four metastatic lymph nodes (M: 7A, 1A, 8A, and 9A) and two non-metastatic lymph nodes (NM: 4C and 5C) resected from patients with T2N0 tongue squamous cell carcinomas. Non-supervised hierarchical clustering plotted based on the average ΔCt values. Up-regulated and down-regulated microRNAs are shown as red and blue, respectively. The columns represent samples and the microRNAs differentially expressed are shown in the lines.
Mentions: Comprehensive microRNA profiles were generated for metastatic (n = 4) and non-metastatic (n = 2) lymph nodes from HNSCC patients using a quantitative RT-PCR array platform. From a total of 667 microRNAs, 439 were detected in at least two samples, regardless of the group, thereby serving as the pool of data for further analyses. From those, 61 presented a P <0.05 in the Rank Products test with 47 showing at least two-fold upregulation in all four metastatic samples. A non-supervised hierarchical clustering analysis using the ΔCt values of these 47 microRNAs displayed two distinct clusters formed by metastatic and non-metastatic lymph nodes (Figure 3). Finally, seven microRNAs (miR-200a, miR-200c, miR-203, miR-205, miR-382, miR-628-5p, miR-758), presenting more than 100-fold increment in the expression level in metastatic lymph nodes, were selected for further analyses (Table 1).Figure 3

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