Cancer Res Treat.  2022 Jan;54(1):174-181. 10.4143/crt.2020.749.

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction (Direct RT-LAMP) for the Detection of Lymph Node Metastasis in Early Breast Cancer

• Lee IH
• Jung JH
• Lee SJ
• Lee J
• Park HY
• Park JY
• Park JY
• Jung Jh
• Lee H
• Jeon HS
• Chae YS

• KMID: 2524598
• DOI: http://doi.org/10.4143/crt.2020.749

Abstract

Purpose
Assessing lymph node metastasis, tumor-derived DNA, or tumor-derived RNA has previously been studied in place of immunohistochemical assay. Because a direct reverse transcription loop-mediated isothermal amplification method (direct RT-LAMP) has been previously developed in order to rapidly identify viruses in place of RNA extraction, our team hypothesized that a direct RT-LAMP assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients.
Materials and Methods
A total amount of 92 lymph nodes removed across 40 patients possessing breast cancer were collected at Kyungpook National University Chilgok Hospital between the months of November 2015 and February 2016. All samples were then evaluated and contrasted via both a direct RT-LAMP assay and routine histopathologic examination.
Results
The sensitivity and specificity of the direct RT-LAMP assay were 85.7% and 100%, respectively. The positive predictive value and negative predictive value were 100% and 94.4%, respectively.
Conclusion
Direct RT-LAMP assay is capable of facilitating the detection of sentinel lymph node metastasis within breast cancer patients intraoperatively possessing an excellent sensitivity via a cost-effective and time-saving manner.

Keyword

Breast neoplasms; Lymphatic metastasis; Reverse transcription loop-mediated isothermal amplification

Figure

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  Fig. 1 Flow chart of the study design. LN, lymph node; neg, negative; pos, positive.

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  Fig. 2 The reverse transcription loop-mediated isothermal amplification primers for cytokeratin 19 (CK19) and ACTB.

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  Fig. 3 Schematic procedural diagram of direct reverse transcription loop-mediated isothermal amplification (RT-LAMP). The total reaction time of our direct RT-LAMP assay is about 30–35 minutes. ACTB, β-actin; CK19, cytokeratin 19; NC, negative control.

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  Fig. 4 Sensitivity of reverse transcription loop-mediated isothermal amplification (RT-LAMP). (A) Sensitivity of CK19 in MCF7 cell line by RT-LAMP. (B) RT-LAMP products by 2% agarose gel electrophoresis. Lane 1, 100 ng; lane 2, 10 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6, positive control (ACTB); lane 7, negative control (D.W.); M, 100-bp marker.

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  Fig. 5 Determination of cutoff value for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay between the histopathologically positive and negative lymph nodes (LNs). Using the cutoff value of 1 ng, we achieved 100% matched results between the RT-LAMP assay and histopathological examination. N, negative; NC, negative control; PC, positive control.

Reference

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