Detection of bone marrow micrometastases by reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with early breast cancer

Öz

OBJECTIVES The aim of this study was to determine the feasibility of a molecular assay by investigating cytokeratin 19 (CK-19) and DF3 gene expressions in detection of occult cancer cells in bone marrow in patients with breast cancer. METHODS Transcriptase-polymerase chain reaction (RT-PCR) assays were carried out for the detection of CK19 and DF3 mRNA transcripts in bone marrow samples of 28 clinically axillary negative patients with early breast cancer. RESULTS Median age of patients was 47.5 (range, 32-86). Due to the high false positivity rate of the CK19-RT-PCR assay, and negative findings by the DF3-RT-PCR assay in bone marrow samples of patients with chronic myelogenous leukemia as negative controls, DF3 expression was tested in further RT-PCR assays. E i g hteen patients (64%) were found to harbor DF3-positivity in bone marrow samples. No statistically significant associations could be found between DF3-positivity and other parameters including stage, tumor size (2 cm), axillary involvement, ER/PR positivity, and high histologic or nuclear grade. CONCLUSION Our results suggest that testing DF3 expression by RT-PCR technique seems to be a reliable method in detection of micrometastatic cells in bone marrow in patients with early breast cancer because of its high specificity.

Anahtar Kelimeler

• Breast Neoplasms,Reverse Transcriptase Polymerase Chain Reaction,Bone Marrow Neoplasms,Bone Marrow,Neoplasm Metastasis,Keratin-19,Gene Expression,DF3 breast-carcinoma-associated antigen

Erken evre meme kanserinde revers transkriptaz polimeraz zincir reaksiyonu (RT-PCR) tekniği ile kemik iliği mikrometastazlarının araştırılması

Öz

AMAÇ Meme kanserinde kemik iliği mikrometastazı saptanmasında sitokeratin 19 (CK19) ve DF3 gen ekspresyonlarının araştırıldığı moleküler bir yöntemin etkinliğini değerlendirmektir. GEREÇ VE YÖNTEM Klinik olarak aksilla negatif olan 28 erken evre meme kanserli hastadan alınan kemik iliği aspiratlarında revers transkriptaz polimeraz zincir reaksiyonu (RT-PCR) tekniğiyle CK19 ve DF3 genlerine özgü iki ayrı primer kullanılarak mikrometastatik kanser hücresi araştırıldı. BULGULAR Hastaların ortanca yaşı 47,5'tir (32-86). RT-PCR çalışmalarında negatif kontrol olarak kullanılan kronik miyeloid lösemili kemik iliği örneklerinin keratin 19 ekspresyonun pozitif ve DF3 ekspresyonunun ise negatif bulunması üzerine, sonraki deneylerde DF3 primeri kullanı ldı. Yirmi sekiz hastanın 18'inde (%64) DF3 pozitif olarak saptandı. DF3 pozitifliği ile hastaların evresi, tümör büyüklüğü (< 2 cm ve > 2 cm), aksiller tutulum, ER/PR pozitifliği ve HG veya NG yüksekliği gibi parametreler arasında istatistiksel açıdan anlamlı bir fark bulunamadı. SONUÇ Erken evre meme kanserli hastalarda kemik iliğinde RT-PCR ile DF3 gen ekspresyonunun varlığının araştırılması yoluyla mikrometastaz saptanması tekniğinin, spesifikliğinin ve duyarlılığının yüksek olması nedeniyle etkin bir yöntem olduğu düşünülmektedir.

Anahtar Kelimeler

• Kemik iliği metastazları, meme kanseri, mikrometastaz, revers transkriptaz polimeraz zincir reaksiyonu.

Kaynakça

1. 1. Braun S, Pantel K, Müller P, Janni W, Hepp F, Kentenich CR, et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342(8):525- 33.
2. 2. Gerber B, Krause A, Müller H, Richter D, Reimer T, Makovitzky J, et al. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001;19(4):960-71.
3. 3. Braun S, Cevatli BS, Assemi C, Janni W, Kentenich CR, Schindlbeck C, et al. Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy. J Clin Oncol 2001;19(5):1468-75.
4. 4. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353(8):793-802.
5. 5. Benoy IH, Elst H, Philips M, Wuyts H, Van Dam P, Scharpé S, et al. Prognostic significance of disseminated tumor cells as detected by quantitative real-time reverse-transcriptase polymerase chain reaction in patients with breast cancer. Clin Breast Cancer 2006;7(2):146-52.
6. 6. Fehm T, Braun S, Muller V, Janni W, Gebauer G, Marth C, et al. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 2006;107(5):885-92.
7. 7. Gilbey AM, Burnett D, Coleman RE, Holen I. The detection of circulating breast cancer cells in blood. J Clin Pathol 2004;57(9):903-11.
8. 8. Baker M, Gillanders WE, Mikhitarian K, Mitas M, Cole DJ. The molecular detection of micrometastatic breast cancer. Am J Surg 2003;186(4):351-8.

9. 9. Martin VM, Siewert C, Scharl A, Harms T, Heinze R, Ohl S, et al. Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. Exp Hematol 1998;26(3):252-64.
10. 10.Cabioglu N, Igci A, Yildirim EO, Aktas E, Bilgic S, Yavuz E, et al. An ultrasensitive tumor enriched flowcytometric assay for detection of isolated tumor cells in bone marrow of patients with breast cancer. Am J Surg 2002;184(5):414-7.
11. 11. Ko Y, Grünewald E, Totzke G, Klinz M, Fronhoffs S, Gouni-Berthold I, et al. High percentage of false-positive results of cytokeratin 19 RT-PCR in blood: a model for the analysis of illegitimate gene expression. Oncology 2000;59(1):81-8.
12. 12.JA Lopez Guercero, P. B o l u f e r-Gilabert M. S a n z Alanso, E. B a r r a g a n - G o n z a l e s, et al. Minimal illegiminate levels of cyctokeratin K19 expression in mononuclear blood cells MNBC detected by a RT PCR method. Clinic Chimica Acta 1997;263:105- 11 6 .
13. 13.Berois N, Varangot M, Aizen B, Estrugo R, Zarantonelli L, Fernández P, et al. Molecular detection of cancer cells in bone marrow and peripheral blood of patients with operable breast cancer. Comparison of CK19, MUC1 and CEA using RT-PCR. Eur J Cancer 2000;36(6):717-23.
14. 14.Bosma AJ, Weigelt B, Lambrechts AC, Verhagen OJ, Pruntel R, Hart AA, et al. Detection of circulating breast tumor cells by differential expression of marker genes. Clin Cancer Res 2002;8(6):1871-7.
15. 15.Berois N, Varangot M, Sóñora C, Zarantonelli L, Pressa C, Laviña R, et al. Detection of bone marrowdisseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA. Int J Cancer 2003;103(4):550-5.
16. 16. Kufer P, Zippelius A, Lutterbüse R, Mecklenburg I, Enzmann T, Montag A, et al. Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res 2002;62(1):251-61.
17. 17. Dent GA, Civalier CJ, Brecher ME, Bentley SA. MUC1 expression in hematopoietic tissues. Am J Clin Pathol 1999;111(6):741-7.
18. 18. Silva AL, Tomé MJ, Correia AE, Passos-Coelho JL. Human mammaglobin RT-PCR assay for detection of occult breast cancer cells in hematopoietic products. Ann Oncol 2002;13(3):422-9.
19. 19.Bossolasco P, Ricci C, Farina G, Soligo D, Pedretti D, Scanni A, et al. Detection of micrometastatic cells in breast cancer by RT-pCR for the mammaglobin gene. Cancer Detect Prev 2002;26(1):60-3.
20. 20. Varangot M, Barrios E, Sóñora C, Aizen B, Pressa C, Estrugo R, et al. Clinical evaluation of a panel of mRNA markers in the detection of disseminated tumor cells in patients with operable breast cancer. Oncol Rep 2005;14(2):537-45.
21. 21. Hayes DF, Mesa-Tejada R, Papsidero LD, Croghan GA, Korzun AH, Norton L, et al. Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a Cancer and Leukemia Group B study. J Clin Oncol 1991;9(7):1113-23.
22. 22.Ismail MS, Wynendaele W, Aerts JL, Paridaens R, Gaafar R, Shakankiry N, et al. Detection of micrometastatic disease and monitoring of perioperative tumor cell dissemination in primary operable breast cancer patients using real-time quantitative reverse transcription-PCR. Clin Cancer Res 2004;10(1 Pt 1):196-201.
23. 23. Zhong XY, Kaul S, Lin YS, Eichler A, Bastert G. Sensitive detection of micrometastases in bone marrow from patients with breast cancer using immunomagnetic isolation of tumor cells in combination with reverse transcriptase/polymerase chain reaction for cytokeratin-19. J Cancer Res Clin Oncol 2000;126(4):212-8.
24. 24. Slade MJ, Smith BM, Sinnett HD, Cross NC, Coombes RC. Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 1999;17(3):870-9.
25. 25. Benoy IH, Salgado R, Elst H, Dam PV, Weyler J, Marck EV, et al. Relative microvessel area of the primary tumor, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinacally non-metastatic breast cancer. Breast Cancer Res 2005;7:210-9.
26. 26. Tokunaga E, Ishida M, Kimura Y, Maehara Y. Correlation with bone metastasis and high expression of CK 19 mRNA measured by quantitative RT-PCR in the bone marrow of breast cancer patients. Breast J 2003;9(5):440-2.
27. 27. Aerts J, Wynendaele W, Paridaens R, Christiaens MR, van den Bogaert W, van Oosterom AT, et al. A realtime quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood. Ann Oncol 2001;12(1):39-46.
28. 28. Lambrechts AC, Bosma AJ, Klaver SG, Top B, Perebolte L, van’ t Veer LJ, et al. Comparison of i m m u n o c y t o c h e m i s t r y, reverse transcriptase polymerase chain reaction, and nucleic acid sequencebased amplification for the detection of circulating breast cancer cells. Breast Cancer Res Tr e a t 1999;56(3):219-31.
29. 29.Balducci E, Azzarello G, Valori L, To ffolatti L, Bolgan L, Valenti MT, et al. A new nested primer pair improves the specificity of CK-19 mRNA detection by RT-PCR in occult breast cancer cells. Int J Biol Markers 2005;20(1):28-33.
30. 30.Brown DC, Purushotham AD, Birnie GD, George WD. Detection of intraoperative tumor cell dissemination in patients with breast cancer by use of reverse transcription and polymerase chain reaction. Surgery 1995;117(1):95-101.
31. 31. Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, et al. Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol 2002;20(17):3628-36.
32. 32. Traweek ST, Liu J, Battifora H. Keratin gene expression in non-epithelial tissues. Detection with polymerase chain reaction. Am J Pathol 1993;142(4):1111-8.
33. 33. Datta YH, Adams PT, Drobyski WR, Ethier SP, Terry VH, Roth MS. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994;12(3):475-82.
34. 34. Moll R, Löwe A, Laufer J, Franke WW. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992;140(2):427-47.
35. 35.Ruud P, Fodstad O, Hovig E. Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells. Int J Cancer 1999;80(1):119-25.
36. 36.Jung YS, Lee KJ, Kim HJ, Yim HE, Park JS, Soh EY, et al. Clinical significance of bone marrow micrometastasis detected by nested rt-PCR for keratin-19 in breast cancer patients. Jpn J Clin Oncol 2003;33(4):167-72.
37. 37. Masuda TA, Kataoka A, Ohno S, Murakami S, Mimori K, Utsunomiya T, et al. Detection of occult cancer cells in peripheral blood and bone marrow by quantitative RT-PCR assay for cytokeratin-7 in breast cancer patients. Int J Oncol 2005;26(3):721-30.