Basic Aspect: Methodology

  • Shuhei Ito
  • Takaaki Masuda
  • Yosuke Kuroda
  • Hidetoshi Eguchi
  • Koshi MimoriEmail author


Initially, micrometastases in the regional lymph nodes (lymph node [LN] micrometastases; LNMs) were detected by immunohistochemical analysis of epithelial markers, and the prognostic significance of LNMs has been a major focus for many years. In addition, recent technological innovations have enabled us to detect LNMs more accurately, with some techniques even showing promising applications in the clinical setting. In this chapter, we will review the methodology to detect LNMs, as follows. First, we will discuss the histopathological method to detect LNMs including conventional hematoxylin and eosin (H&E) staining and immunohistochemical analysis. Next, we will describe methods for detection of cancer-associated mRNA and review quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method, which are currently being used in the clinical setting. Third, genetic or epigenetic techniques to detect LNMs will be discussed, and we will introduce methods to detect LNMs by cancer-specific events, such as mutations and methylation. Finally, we will describe future perspectives, such as potential discoveries related to LNMs, with a specific focus on the development of new strategies for detecting LNMs.


Immunohistochemistry mRNA expression Mutation Methylation Visualization Malignant potential Host factor 


  1. 1.
    Sobin L, Gospodarowicz M, Wittekind C. TNM classification of malignant tumours. 7th ed. Hoboken, NJ: Wiley-Blackwell; 2009.Google Scholar
  2. 2.
    Isozaki H, Okajima K, Fujii K. Histological evaluation of lymph node metastasis on serial sectioning in gastric cancer with radical lymphadenectomy. Hepato-Gastroenterology. 1997;44:1133–6.PubMedGoogle Scholar
  3. 3.
    Natsugoe S, Aikou T, Shimada M, Yoshinaka H, Takao S, Shimazu H, et al. Occult lymph node metastasis in gastric cancer with submucosal invasion. Surg Today. 1994;24:870–5.CrossRefGoogle Scholar
  4. 4.
    Noura S, Yamamoto H, Miyake Y, Kim B, Takayama O, Seshimo I, et al. Immunohistochemical assessment of localization and frequency of micrometastases in lymph nodes of colorectal cancer. Clin Cancer Res. 2002;8:759–67.PubMedGoogle Scholar
  5. 5.
    Maehara Y, Oshiro T, Endo K, Baba H, Oda S, Ichiyoshi Y, et al. Clinical significance of occult micrometastasis lymph nodes from patients with early gastric cancer who died of recurrence. Surgery. 1996;119:397–402.CrossRefGoogle Scholar
  6. 6.
    Cai J, Ikeguchi M, Maeta M, Kaibara N. Micrometastasis in lymph nodes and microinvasion of the muscularis propria in primary lesions of submucosal gastric cancer. Surgery. 2000;127:32–9.CrossRefGoogle Scholar
  7. 7.
    Lee E, Chae Y, Kim I, Choi J, Yeom B, Leong AS. Prognostic relevance of immunohistochemically detected lymph node micrometastasis in patients with gastric carcinoma. Cancer. 2002;94:2867–73.CrossRefGoogle Scholar
  8. 8.
    Matsumoto M, Natsugoe S, Ishigami S, Uenosono Y, Takao S, Aikou T. Rapid immunohistochemical detection of lymph node micrometastasis during operation for upper gastrointestinal carcinoma. Br J Surg. 2003;90:563–6.CrossRefGoogle Scholar
  9. 9.
    Sanei MH, Tabatabie SA, Hashemi SM, Cherei A, Mahzouni P, Sanei B. Comparing the efficacy of routine H&E staining and cytokeratin immunohistochemical staining in detection of micro-metastasis on serial sections of dye-mapped sentinel lymph nodes in colorectal carcinoma. Adv Biomed Res. 2016;5:13.CrossRefGoogle Scholar
  10. 10.
    Kumagai K, Yamamoto N, Miyashiro I, Tomita Y, Katai H, Kushima R, et al. Multicenter study evaluating the clinical performance of the OSNA assay for the molecular detection of lymph node metastases in gastric cancer patients. Gastric Cancer. 2014;17:273–80.CrossRefGoogle Scholar
  11. 11.
    Natsugoe S, Arigami T, Uenosono Y, Yanagita S, Nakajo A, Matsumoto M, et al. Lymph node micrometastasis in gastrointestinal tract cancer--a clinical aspect. Int J Clin Oncol. 2013;18:752–61.CrossRefGoogle Scholar
  12. 12.
    Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005;5:209–19.CrossRefGoogle Scholar
  13. 13.
    Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1988;16:11141–56.CrossRefGoogle Scholar
  14. 14.
    Keilholz U, Willhauck M, Rimoldi D, Brasseur F, Dummer W, Rass K, et al. Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: a quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer. 1998;34:750–3.CrossRefGoogle Scholar
  15. 15.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63.CrossRefGoogle Scholar
  16. 16.
    Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama F, et al. One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Cancer Res. 2007;13:4807–16.CrossRefGoogle Scholar
  17. 17.
    Tamaki Y, Akiyama F, Iwase T, Kaneko T, Tsuda H, Sato K, et al. 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. 2009;15:2879–84.CrossRefGoogle Scholar
  18. 18.
    Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009;15:62–9.CrossRefGoogle Scholar
  19. 19.
    Snook KL, Layer GT, Jackson PA, de Vries CS, Shousha S, Sinnett HD, et al. Multicentre evaluation of intraoperative molecular analysis of sentinel lymph nodes in breast carcinoma. Br J Surg. 2011;98:527–35.CrossRefGoogle Scholar
  20. 20.
    Cserni G. Intraoperative analysis of sentinel lymph nodes in breast cancer by one-step nucleic acid amplification. J Clin Pathol. 2012;65:193–9.CrossRefGoogle Scholar
  21. 21.
    Tamaki Y. One-step nucleic acid amplification (OSNA): where do we go with it? Int J Clin Oncol. 2017;22(1):3–10.CrossRefGoogle Scholar
  22. 22.
    Rahbari NN, Bork U, Motschall E, Thorlund K, Buchler MW, Koch M, et al. Molecular detection of tumor cells in regional lymph nodes is associated with disease recurrence and poor survival in node-negative colorectal cancer: a systematic review and meta-analysis. J Clin Oncol. 2012;30:60–70.CrossRefGoogle Scholar
  23. 23.
    Losi L, Benhattar J, Costa J. Stability of K-ras mutations throughout the natural history of human colorectal cancer. Eur J Cancer. 1992;28A:1115–20.CrossRefGoogle Scholar
  24. 24.
    Hayashi N, Arakawa H, Nagase H, Yanagisawa A, Kato Y, Ohta H, et al. Genetic diagnosis identifies occult lymph node metastases undetectable by the histopathological method. Cancer Res. 1994;54:3853–6.PubMedGoogle Scholar
  25. 25.
    Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998;72:141–96.CrossRefGoogle Scholar
  26. 26.
    Sanchez-Cespedes M, Esteller M, Hibi K, Cope FO, Westra WH, Piantadosi S, et al. Molecular detection of neoplastic cells in lymph nodes of metastatic colorectal cancer patients predicts recurrence. Clin Cancer Res. 1999;5:2450–4.PubMedGoogle Scholar
  27. 27.
    Urano Y, Sakabe M, Kosaka N, Ogawa M, Mitsunaga M, Asanuma D, et al. Rapid cancer detection by topically spraying a gamma-glutamyltranspeptidase-activated fluorescent probe. Sci Transl Med. 2011;3:110ra9.CrossRefGoogle Scholar
  28. 28.
    Ueo H, Shinden Y, Tobo T, Gamachi A, Udo M, Komatsu H, et al. Rapid intraoperative visualization of breast lesions with gamma-glutamyl hydroxymethyl rhodamine green. Sci Rep. 2015;5:12080.CrossRefGoogle Scholar
  29. 29.
    Shinden Y, Ueo H, Tobo T, Gamachi A, Utou M, Komatsu H, et al. Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with gamma-glutamyl hydroxymethyl rhodamine green. Sci Rep. 2016;6:27525.CrossRefGoogle Scholar
  30. 30.
    Yano S, Takehara K, Miwa S, Kishimoto H, Hiroshima Y, Murakami T, et al. Improved resection and outcome of colon-cancer liver metastasis with fluorescence-guided surgery using in situ GFP labeling with a telomerase-dependent adenovirus in an orthotopic mouse model. PLoS One. 2016;11:e0148760.CrossRefGoogle Scholar
  31. 31.
    Shigeyasu K, Tazawa H, Hashimoto Y, Mori Y, Nishizaki M, Kishimoto H, et al. Fluorescence virus-guided capturing system of human colorectal circulating tumour cells for non-invasive companion diagnostics. Gut. 2015;64:627–35.CrossRefGoogle Scholar
  32. 32.
    Ito H, Inoue H, Kimura S, Ohmori T, Ishikawa F, Gohda K, et al. Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: a prospective study. Int J Oncol. 2014;45:227–34.CrossRefGoogle Scholar
  33. 33.
    Karaman S, Detmar M. Mechanisms of lymphatic metastasis. J Clin Invest. 2014;124:922–8.CrossRefGoogle Scholar
  34. 34.
    Hirakawa S, Kodama S, Kunstfeld R, Kajiya K, Brown LF, Detmar M. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J Exp Med. 2005;201:1089–99.CrossRefGoogle Scholar
  35. 35.
    Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol. 1999;144:789–801.CrossRefGoogle Scholar
  36. 36.
    Mumprecht V, Honer M, Vigl B, Proulx ST, Trachsel E, Kaspar M, et al. In vivo imaging of inflammation- and tumor-induced lymph node lymphangiogenesis by immuno-positron emission tomography. Cancer Res. 2010;70:8842–51.CrossRefGoogle Scholar
  37. 37.
    Proulx ST, Luciani P, Derzsi S, Rinderknecht M, Mumprecht V, Leroux JC, et al. Quantitative imaging of lymphatic function with liposomal indocyanine green. Cancer Res. 2010;70:7053–62.CrossRefGoogle Scholar
  38. 38.
    Proulx ST, Luciani P, Christiansen A, Karaman S, Blum KS, Rinderknecht M, et al. Use of a PEG-conjugated bright near-infrared dye for functional imaging of rerouting of tumor lymphatic drainage after sentinel lymph node metastasis. Biomaterials. 2013;34:5128–37.CrossRefGoogle Scholar
  39. 39.
    Terashita K, Chuma M, Hatanaka Y, Hatanaka K, Mitsuhashi T, Yokoo H, et al. ZEB1 expression is associated with prognosis of intrahepatic cholangiocarcinoma. J Clin Pathol. 2016;69:593–9.CrossRefGoogle Scholar
  40. 40.
    Ma H, Gao L, Li S, Qin J, Chen L, Liu X, et al. CCR7 enhances TGF-beta1-induced epithelial-mesenchymal transition and is associated with lymph node metastasis and poor overall survival in gastric cancer. Oncotarget. 2015;6:24348–60.PubMedPubMedCentralGoogle Scholar
  41. 41.
    Zhang G, Zhou H, Xiao H, Liu Z, Tian H, Zhou T. MicroRNA-92a functions as an oncogene in colorectal cancer by targeting PTEN. Dig Dis Sci. 2014;59:98–107.CrossRefGoogle Scholar
  42. 42.
    Zhang F, Luo Y, Shao Z, Xu L, Liu X, Niu Y, et al. MicroRNA-187, a downstream effector of TGFbeta pathway, suppresses Smad-mediated epithelial-mesenchymal transition in colorectal cancer. Cancer Lett. 2016;373:203–13.CrossRefGoogle Scholar
  43. 43.
    Taniguchi H, Moriya C, Igarashi H, Saitoh A, Yamamoto H, Adachi Y, et al. Cancer stem cells in human gastrointestinal cancer. Cancer Sci. 2016;107:1556.CrossRefGoogle Scholar
  44. 44.
    Kim M, Koh YJ, Kim KE, Koh BI, Nam DH, Alitalo K, et al. CXCR4 signaling regulates metastasis of chemoresistant melanoma cells by a lymphatic metastatic niche. Cancer Res. 2010;70:10411–21.CrossRefGoogle Scholar
  45. 45.
    Masuda T, Hayashi N, Iguchi T, Ito S, Eguchi H, Mimori K. Clinical and biological significance of circulating tumor cells in cancer. Mol Oncol. 2016;10:408–17.CrossRefGoogle Scholar
  46. 46.
    Mashino K, Sadanaga N, Yamaguchi H, Tanaka F, Ohta M, Shibuta K, et al. Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res. 2002;62:2937–41.PubMedGoogle Scholar
  47. 47.
    Gunther K, Leier J, Henning G, Dimmler A, Weissbach R, Hohenberger W, et al. Prediction of lymph node metastasis in colorectal carcinoma by expression of chemokine receptor CCR7. Int J Cancer. 2005;116:726–33.CrossRefGoogle Scholar
  48. 48.
    Yumimoto K, Akiyoshi S, Ueo H, Sagara Y, Onoyama I, Ueo H, et al. F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner. J Clin Invest. 2015;125:621–35.CrossRefGoogle Scholar
  49. 49.
    Das S, Sarrou E, Podgrabinska S, Cassella M, Mungamuri SK, Feirt N, et al. Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses. J Exp Med. 2013;210:1509–28.CrossRefGoogle Scholar
  50. 50.
    Tewalt EF, Cohen JN, Rouhani SJ, Engelhard VH. Lymphatic endothelial cells – key players in regulation of tolerance and immunity. Front Immunol. 2012;3:305.CrossRefGoogle Scholar
  51. 51.
    Tewalt EF, Cohen JN, Rouhani SJ, Guidi CJ, Qiao H, Fahl SP, et al. Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells. Blood. 2012;120:4772–82.CrossRefGoogle Scholar
  52. 52.
    Shields JD, Kourtis IC, Tomei AA, Roberts JM, Swartz MA. Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21. Science. 2010;328:749–52.CrossRefGoogle Scholar
  53. 53.
    Lund AW, Duraes FV, Hirosue S, Raghavan VR, Nembrini C, Thomas SN, et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics. Cell Rep. 2012;1:191–9.CrossRefGoogle Scholar
  54. 54.
    Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res. 2011;71:3792–801.CrossRefGoogle Scholar
  55. 55.
    Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–8.CrossRefGoogle Scholar
  56. 56.
    Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q, Bos PD, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature. 2008;451:147–52.CrossRefGoogle Scholar
  57. 57.
    Zhang FF, Luo YH, Wang H, Zhao L. Metastasis-associated long noncoding RNAs in gastrointestinal cancer: implications for novel biomarkers and therapeutic targets. World J Gastroenterol. 2016;22:8735–49.CrossRefGoogle Scholar
  58. 58.
    Burrell RA, McGranahan N, Bartek J, Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature. 2013;501:338–45.CrossRefGoogle Scholar
  59. 59.
    Uchi R, Takahashi Y, Niida A, Shimamura T, Hirata H, Sugimachi K, et al. Integrated multiregional analysis proposing a new model of colorectal cancer evolution. PLoS Genet. 2016;12:e1005778.CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  • Shuhei Ito
    • 1
  • Takaaki Masuda
    • 1
  • Yosuke Kuroda
    • 1
  • Hidetoshi Eguchi
    • 1
  • Koshi Mimori
    • 1
    Email author
  1. 1.Department of SurgeryKyushu University Beppu HospitalBeppuJapan

Personalised recommendations