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. 1999 Aug;80(12):2019–2024. doi: 10.1038/sj.bjc.6690636

Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer

A E H Merrie 1, K Yun 2, J Gunn 1, L V Phillips 1, J L McCall 3
PMCID: PMC2363147  PMID: 10471055

Abstract

We have developed sensitive assays for cytokeratin (K) 8, 16, 19, stromelysin 3 (ST3), MUC1 and maspin mRNAs using reverse transcription polymerase chain reaction (RT-PCR) and used these to assess lymph node status in patients undergoing surgery for breast cancer. In addition the RT-PCR assays were tested against lymph nodes from non-cancer patients to determine their specificity. Despite high sensitivity RT-PCR assays for K8, K16, K19, ST3 and maspin were not found to be useful as markers of submicroscopic disease as transcripts of these genes were detected in the great majority of control lymph nodes tested. Expression of MUC1 was also not found to be useful as it was both insensitive and non-specific. The importance of assessing potential markers against an adequately sized control population is demonstrated, as failure to do so can lead to erroneous conclusions. © 1999 Cancer Research Campaign

Keywords: breast cancer, lymph node metastases, RT-PCR, molecular markers

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Selected References

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  1. Abe M., Kufe D. Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):282–286. doi: 10.1073/pnas.90.1.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams M. D., Kerlavage A. R., Fleischmann R. D., Fuldner R. A., Bult C. J., Lee N. H., Kirkness E. F., Weinstock K. G., Gocayne J. D., White O. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature. 1995 Sep 28;377(6547 Suppl):3–174. [PubMed] [Google Scholar]
  3. Ahmad A., Hanby A., Dublin E., Poulsom R., Smith P., Barnes D., Rubens R., Anglard P., Hart I. Stromelysin 3: an independent prognostic factor for relapse-free survival in node-positive breast cancer and demonstration of novel breast carcinoma cell expression. Am J Pathol. 1998 Mar;152(3):721–728. [PMC free article] [PubMed] [Google Scholar]
  4. BLOOM H. J., RICHARDSON W. W. Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957 Sep;11(3):359–377. doi: 10.1038/bjc.1957.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bader B. L., Jahn L., Franke W. W. Low level expression of cytokeratins 8, 18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene. Eur J Cell Biol. 1988 Dec;47(2):300–319. [PubMed] [Google Scholar]
  6. Brown D. C., Purushotham A. D., Birnie G. D., George W. D. Detection of intraoperative tumor cell dissemination in patients with breast cancer by use of reverse transcription and polymerase chain reaction. Surgery. 1995 Jan;117(1):95–101. doi: 10.1016/s0039-6060(05)80235-1. [DOI] [PubMed] [Google Scholar]
  7. Burchill S. A., Bradbury M. F., Pittman K., Southgate J., Smith B., Selby P. Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br J Cancer. 1995 Feb;71(2):278–281. doi: 10.1038/bjc.1995.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Datta Y. H., Adams P. T., Drobyski W. R., Ethier S. P., Terry V. H., Roth M. S. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol. 1994 Mar;12(3):475–482. doi: 10.1200/JCO.1994.12.3.475. [DOI] [PubMed] [Google Scholar]
  10. Denis M. G., Lipart C., Leborgne J., LeHur P. A., Galmiche J. P., Denis M., Ruud E., Truchaud A., Lustenberger P. Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients. Int J Cancer. 1997 Oct 21;74(5):540–544. doi: 10.1002/(sici)1097-0215(19971021)74:5<540::aid-ijc11>3.0.co;2-a. [DOI] [PubMed] [Google Scholar]
  11. Dingemans A. M., Brakenhoff R. H., Postmus P. E., Giaccone G. Detection of cytokeratin-19 transcripts by reverse transcriptase-polymerase chain reaction in lung cancer cell lines and blood of lung cancer patients. Lab Invest. 1997 Sep;77(3):213–220. [PubMed] [Google Scholar]
  12. Eltahir E. M., Mallinson D. S., Birnie G. D., Hagan C., George W. D., Purushotham A. D. Putative markers for the detection of breast carcinoma cells in blood. Br J Cancer. 1998 Apr;77(8):1203–1207. doi: 10.1038/bjc.1998.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ghossein R. A., Rosai J. Polymerase chain reaction in the detection of micrometastases and circulating tumor cells. Cancer. 1996 Jul 1;78(1):10–16. doi: 10.1002/(SICI)1097-0142(19960701)78:1<10::AID-CNCR3>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  14. Gunn J., McCall J. L., Yun K., Wright P. A. Detection of micrometastases in colorectal cancer patients by K19 and K20 reverse-transcription polymerase chain reaction. Lab Invest. 1996 Oct;75(4):611–616. [PubMed] [Google Scholar]
  15. Hoon D. S., Doi F., Giuliano A. E., Schmid P., Conrad A. J. The detection of breast carcinoma micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Cancer. 1995 Aug 1;76(3):533–535. doi: 10.1002/1097-0142(19950801)76:3<533::aid-cncr2820760330>3.0.co;2-t. [DOI] [PubMed] [Google Scholar]
  16. Jonas S., Windeatt S., O-Boateng A., Fordy C., Allen-Mersh T. G. Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction. Gut. 1996 Nov;39(5):717–721. doi: 10.1136/gut.39.5.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krismann M., Todt B., Schröder J., Gareis D., Müller K. M., Seeber S., Schütte J. Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. J Clin Oncol. 1995 Nov;13(11):2769–2775. doi: 10.1200/JCO.1995.13.11.2769. [DOI] [PubMed] [Google Scholar]
  18. Liefers G. J., Cleton-Jansen A. M., van de Velde C. J., Hermans J., van Krieken J. H., Cornelisse C. J., Tollenaar R. A. Micrometastases and survival in stage II colorectal cancer. N Engl J Med. 1998 Jul 23;339(4):223–228. doi: 10.1056/NEJM199807233390403. [DOI] [PubMed] [Google Scholar]
  19. Lockett M. A., Baron P. L., O'Brien P. H., Elliott B. M., Robison J. G., Maitre N., Metcalf J. S., Cole D. J. Detection of occult breast cancer micrometastases in axillary lymph nodes using a multimarker reverse transcriptase-polymerase chain reaction panel. J Am Coll Surg. 1998 Jul;187(1):9–16. doi: 10.1016/s1072-7515(98)00130-6. [DOI] [PubMed] [Google Scholar]
  20. Luppi M., Morselli M., Bandieri E., Federico M., Marasca R., Barozzi P., Ferrari M. G., Savarino M., Frassoldati A., Torelli G. Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene. Ann Oncol. 1996 Aug;7(6):619–624. doi: 10.1093/oxfordjournals.annonc.a010680. [DOI] [PubMed] [Google Scholar]
  21. Matsumura Y., Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet. 1992 Oct 31;340(8827):1053–1058. doi: 10.1016/0140-6736(92)93077-z. [DOI] [PubMed] [Google Scholar]
  22. Merrie A. E., Yun K., McCall J. L. Detection of carcinoembryonic antigen messenger RNA in lymph nodes from patients with colorectal cancer. N Engl J Med. 1998 Nov 26;339(22):1642–1644. doi: 10.1056/NEJM199811263392215. [DOI] [PubMed] [Google Scholar]
  23. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  24. Mori M., Mimori K., Inoue H., Barnard G. F., Tsuji K., Nanbara S., Ueo H., Akiyoshi T. Detection of cancer micrometastases in lymph nodes by reverse transcriptase-polymerase chain reaction. Cancer Res. 1995 Aug 1;55(15):3417–3420. [PubMed] [Google Scholar]
  25. Noguchi S., Aihara T., Motomura K., Inaji H., Imaoka S., Koyama H. Detection of breast cancer micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Comparison between MUC1 mRNA and keratin 19 mRNA amplification. Am J Pathol. 1996 Feb;148(2):649–656. [PMC free article] [PubMed] [Google Scholar]
  26. Noguchi S., Aihara T., Motomura K., Inaji H., Imaoka S., Koyama H. Histologic characteristics of breast cancers with occult lymph node metastases detected by keratin 19 mRNA reverse transcriptase-polymerase chain reaction. Cancer. 1996 Sep 15;78(6):1235–1240. doi: 10.1002/(SICI)1097-0142(19960915)78:6<1235::AID-CNCR10>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  27. Noguchi S., Aihara T., Nakamori S., Motomura K., Inaji H., Imaoka S., Koyama H. The detection of breast carcinoma micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Cancer. 1994 Sep 1;74(5):1595–1600. doi: 10.1002/1097-0142(19940901)74:5<1595::aid-cncr2820740516>3.0.co;2-l. [DOI] [PubMed] [Google Scholar]
  28. Schoenfeld A., Kruger K. H., Gomm J., Sinnett H. D., Gazet J. C., Sacks N., Bender H. G., Luqmani Y., Coombes R. C. The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer. 1997 May;33(6):854–861. doi: 10.1016/s0959-8049(97)00014-2. [DOI] [PubMed] [Google Scholar]
  29. Schoenfeld A., Luqmani Y., Sinnett H. D., Shousha S., Coombes R. C. Keratin 19 mRNA measurement to detect micrometastases in lymph nodes in breast cancer patients. Br J Cancer. 1996 Nov;74(10):1639–1642. doi: 10.1038/bjc.1996.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schoenfeld A., Luqmani Y., Smith D., O'Reilly S., Shousha S., Sinnett H. D., Coombes R. C. Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction. Cancer Res. 1994 Jun 1;54(11):2986–2990. [PubMed] [Google Scholar]
  31. Traweek S. T., Liu J., Battifora H. Keratin gene expression in non-epithelial tissues. Detection with polymerase chain reaction. Am J Pathol. 1993 Apr;142(4):1111–1118. [PMC free article] [PubMed] [Google Scholar]
  32. Yun K., Gunn J., Merrie A. E., Phillips L. V., McCall J. L. Keratin 19 mRNA is detectable by RT-PCR in lymph nodes of patients without breast cancer. Br J Cancer. 1997;76(8):1112–1113. doi: 10.1038/bjc.1997.516. [DOI] [PMC free article] [PubMed] [Google Scholar]

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