An intra-operative RT-LAMP method allows rapid and reliable detection of sentinel lymph node metastasis in breast cancer patients
- 479 Downloads
The rapid determination of metastasis in sentinel lymph nodes (SLNs) of breast cancer patients plays a significant role in the selection of a surgery strategy. Although a previous one-step nucleic acid amplification assay that uses reverse-transcription (RT) loop-mediated isothermal amplification (LAMP) has showed specific advantages over traditional pathological examination, its target marker requires optimisation. In addition to epithelial-specific CK19, the internal control gene PBGD and the breast-specific PIP were included in the new method. After the RT-LAMP primers were designed and verified using a cell line, the performance of our method was evaluated by comparing it with the corresponding result of the Food and Drug Administration approved breast lymph node (BLN) assay and routine pathological examination. One hundred and seventy-four valid SLN samples from 101 patients were collected from five hospitals. The threshold of reaction time for CK19, PIP and PBGD was defined as 16, 20 and 20 min, respectively. Compared with the BLN assay, the concordance rate of our method was 95.4 % (166/174). Statistical analysis revealed that the two methods are consistent (kappa = 0.890, P < 0.001). When compared with pathological examination, the performance of our method (sensitivity = 81.3 %, specificity = 89.7 %, kappa = 0.691, P < 0.001) was similar to that of the BLN assay (sensitivity = 87.5 %, specificity = 84.9 %, kappa = 0.668, P < 0.001). This result demonstrates the potential usefulness of our method in clinical practice. In conclusion, we preliminarily established an intra-operative diagnostic method that assimilates the merits of previous assays. In contrast with the BLN assay and pathological examination, our method can be completed in 30 min and shows high sensitivity, specificity and consistency, which we consider as promising for clinical application.
KeywordsBreast cancer Sentinel lymph node (SLN) Intra-operative diagnostic method Reverse-transcription loop-mediated isothermal amplification (RT-LAMP)
We gratefully acknowledge the excellent assistance of Jia Mi and Fengjing Li (from Beijing Lanpu Bio-tech Co., Ltd.) in design of LAMP primer. The study was supported by the Major Special Project of Beijing Municipal Science and Technology Commission (D09050703570905).
Conflict of interest
The authors declare that they have no competing interests.
- 1.Halsted WS (1984) The results of operations for the cure of cancer of the breast performed at the Johns Hopkins Hospital from June, 1889, to January, 1894. Ann Surg 20(5):497–555Google Scholar
- 10.Lyman GH, Temin S, Edge SB, Newman LA, Turner RR, Weaver DL, Benson AB 3rd, Bosserman LD, Burstein HJ, Cody H 3rd, Hayman J, Perkins CL, Podoloff DA, Giuliano AE (2014) Sentinel lymph node biopsy for patients with early-stage breast cancer: American Society of Clinical Oncology clinical practice guideline update. J Clin Oncol 32(13):1365–1383CrossRefPubMedGoogle Scholar
- 11.National Comprehensive Cancer Network (NCCN) Clinical practice guidelines in oncology. Breast Cancer Version 3.2014Google Scholar
- 12.Cserni G, Amendoeira I, Apostolikas N, Bellocq JP, Bianchi S, Boecker W, Borisch B, Connolly CE, Decker T, Dervan P, Drijkoningen M, Ellis IO, Elston CW, Eusebi V, Faverly D, Heikkila P, Holland R, Kerner H, Kulka J, Jacquemier J, Lacerda M, Martinez-Penuela J, De Miguel C, Peterse JL, Rank F, Regitnig P, Reiner A, Sapino A, Sigal-Zafrani B, Tanous AM, Thorstenson S, Zozaya E, Fejes G, Wells CA (2004) Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European working group for breast screening pathology. J Clin Pathol 57(7):695–701CrossRefPubMedCentralPubMedGoogle Scholar
- 15.Blumencranz P, Whitworth PW, Deck K, Rosenberg A, Reintgen D, Beitsch P, Chagpar A, Julian T, Saha S, Mamounas E, Giuliano A, Simmons R (2007) Scientific impact recognition award. Sentinel node staging for breast cancer: intraoperative molecular pathology overcomes conventional histologic sampling errors. Am J Surg 194(4):426–432CrossRefPubMedGoogle Scholar
- 16.Viale G, Dell’Orto P, Biasi MO, Stufano V, De Brito Lima LN, Paganelli G, Maisonneuve P, Vargo JM, Green G, Cao W, Swijter A, Mazzarol G (2008) Comparative evaluation of an extensive histopathologic examination and a real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 on axillary sentinel lymph nodes of breast carcinoma patients. Ann Surg 247(1):136–142CrossRefPubMedGoogle Scholar
- 17.Julian TB, Blumencranz P, Deck K, Whitworth P, Berry DA, Berry SM, Rosenberg A, Chagpar AB, Reintgen D, Beitsch P, Simmons R, Saha S, Mamounas EP, Giuliano A (2008) Novel intraoperative molecular test for sentinel lymph node metastases in patients with early-stage breast cancer. J Clin Oncol 26(20):3338–3345CrossRefPubMedGoogle Scholar
- 20.Hughes SJ, Xi L, Raja S, Gooding W, Cole DJ, Gillanders WE, Mikhitarian K, McCarty K, Silver S, Ching J, McMillan W, Luketich JD, Godfrey TE (2006) A rapid, fully automated, molecular-based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer. Ann Surg 243(3):389–398CrossRefPubMedCentralPubMedGoogle Scholar
- 21.Hughes SJ, Xi L, Gooding WE, Cole DJ, Mitas M, Metcalf J, Bhargava R, Dabbs D, Ching J, Kozma L, McMillan W, Godfrey TE (2009) A quantitative reverse transcription-PCR assay for rapid, automated analysis of breast cancer sentinel lymph nodes. J Mol Diagn 11(6):576–582CrossRefPubMedCentralPubMedGoogle Scholar
- 22.Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama F, Kato Y, Tsuda H, Ueda S, Sato K, Tamaki Y, Noguchi S, Kataoka TR, Nakajima H, Komoike Y, Inaji H, Tsugawa K, Suzuki K, Nakamura S, Daitoh M, Otomo Y, Matsuura N (2007) One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Cancer Res 13(16):4807–4816CrossRefPubMedGoogle Scholar
- 25.Tamaki Y, Akiyama F, Iwase T, Kaneko T, Tsuda H, Sato K, Ueda S, Mano M, Masuda N, Takeda M, Tsujimoto M, Yoshidome K, Inaji H, Nakajima H, Komoike Y, Kataoka TR, Nakamura S, Suzuki K, Tsugawa K, Wakasa K, Okino T, Kato Y, Noguchi S, Matsuura N (2009) Molecular detection of lymph node metastases in breast cancer patients: results of a multicenter trial using the one-step nucleic acid amplification assay. Clin Cancer Res 15(8):2879–2884CrossRefPubMedGoogle Scholar
- 26.Osako T, Iwase T, Kimura K, Yamashita K, Horii R, Yanagisawa A, Akiyama F (2011) Intraoperative molecular assay for sentinel lymph node metastases in early stage breast cancer: a comparative analysis between one-step nucleic acid amplification whole node assay and routine frozen section histology. Cancer 117(19):4365–4374CrossRefPubMedGoogle Scholar
- 28.Vilardell F, Novell A, Martin J, Santacana M, Velasco A, Díez-Castro MJ, Cuevas D, Panadés MJ, González S, Llombart A, Iglesias E, Matias-Guiu X (2012) Importance of assessing CK19 immunostaining in core biopsies in patients subjected to sentinel node study by OSNA. Virchows Arch 460(6):569–575CrossRefPubMedCentralPubMedGoogle Scholar
- 29.Le Frère-Belda MA, Bats AS, Gillaizeau F, Poulet B, Clough KB, Nos C, Peoc’h M, Seffert P, Bouteille C, Leroux A, Guillemin F, Blanc-Fournier C, Crouet H, Arnould L, Cuisenier J, Penault-Llorca F, Gimbergues P, Jacquemier J, Houvenaeghel G, Chatellier G, Lécuru F (2012) Diagnostic performance of one-step nucleic acid amplification for intraoperative sentinel node metastasis detection in breast cancer patients. Int J Cancer 130(10):2377–2386CrossRefPubMedGoogle Scholar