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The value of bone marrow aspirates culture for the detection of bone marrow micrometastasis in breast cancer
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Biomedicine & Pharmacotherapy 59 (2005) $384-$386 http://france.elsevier.com/direct/BIOPHA/
The value of bone marrow aspirates culture for the detection of bone marrow micrometastasis in breast cancer W.T.Y. Loo, J.H.M. Fong, L. Zhu, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, Universityof Hong Kong Medical Center, PokfulamRoad, Queen Mary Hospital, Hong Kong, China
Abstract
Background. - Detection of micrometastasis is an important problem of clinical significance for a better understanding and control of tumor progression, which will improve patients' survival time. Tumor cells in bone marrow (BM) aspirates are indicative of the general disseminative metastasis in patients with early breast cancer and characterization of breast cancers by various tumor markers which are appropriate for the identification of high risk groups. M a t e r i a l s and methods. - Bone marrow aspirates were obtained from 44 breast cancer patients at the time of surgery. To identify micrometastases in bone marrow, an immunocytochemical assay for epithelial cytokeratin (CK) was performed at the second passage after selective culture. Cytokeratin-positive bone marrow disseminated cancer cells were observed in more than 90% of the patients. This high incidence needs further investigation with bigger sample size to confirm. However, these results indicate that this technique can be used as an early diagnostic technique of bone marrow micrometastasis in the patient with breast cancer thereby promoting the development of therapeutic strategy. High incidences need further investigation with bigger samples to confirm. © 2005 Elsevier SAS. All rights reserved. Keywords: Bone marrow; Micrometastasis; Cytokeratin; Cyclooxygenase 2
1. Introduction The clinical importance of metastasized cells in the bone marrow of breast cancer patients has been demonstrated in several prospective studies, and represents an independent prognostic factor for distant relapse and overall survival [17]. Very little is known about the biological features of the disseminated cancer cells. However, extensive cell culture experiments have shown that cells disseminating into bone marrow have a time-limited proliferative potential [8]. Immunocytochemical (ICC) detection of epithelial cells in the bone marrow of breast cancer patients has been performed with a variety of antibodies thus far in many similar studies [9-12]. Cytokeratin is intermediate filament which present mainly in epithelium. Currently available data suggest that CK-positive cells in bone marrow aspirates
*
Corresponding author.
E-mail address: [email protected] (L.W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
of cancer patients represent a selected, but nevertheless heterogeneous population of dormant (G0-phase) cancer cells [13]. The aim of the current study was to culture micrometastatic tumor cells present in bone marrow of patients with invasive ductal carcinoma and to thereby establish immortalized cell lines. The established system will allow an in-depth molecular analysis of human micrometastatic cancer cells and could become a useful source for the identification of high risk groups. 2. Material and methods 2.1. Cell culture
Bone marrow aspirates anterior superior iliac spine (ASIS) from 44 patients diagnosed with invasive ductal carcinoma were collected in EDTA tubes given their consent. Once they were transferred to the laboratory, the
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $384-$386
red blood cells of the specimens were lyzed with cell lysis buffer for 5 min before being spun down. The cell pellet containing the tumor cells was resuspended and distributed into a plastic 25 cm 2 cell culture flask (Coming Glass Works, Coming, NY, USA), with high-glucose Dulbecco's modified eagle medium (D-MEM) with 15% fetal bovine serum (FBS), 15 mM HEPES buffer, L-glutamine, pyridoxine hydrochloride, penicillin (100 U/ml), streptomycin (100 ~tg/ml) and insulin (4 ~tg/ml) (Invitrogen Corporation, California, USA). The culture flask was incubated at 37 °C in 95% air and 5% CO 2 for 24 h. All culture medium was removed and replaced with fresh medium every 3 days. 2.2. I m m u n o c y t o c h e m i c a l s t u d i e s
Various antigens of monolayers of cells cultured on 4-well SonicSeal Slide (NUNC, USA) were immunocytochemically tested with mouse monoclonal antibody cytokeratin (Dako Corporation, USA) after five passages. This was performed by using a SuperPicture kit (Zymed Laboratories Inc, USA). Cells were fixed in 4% paraformaldehyde and incubated with primary antibodies in a ratio of 1:100 at 4 °C overnight. Staining was performed using DAB substrate-chromogen solution accordingly. Cells were counterstained with hematoxylin and mounted in Permount. The intensity of the stain was graded as negative (-), weak (+), or strong (++), depending on the number of cells stained and the thickness and darkness of the DAB precipitate. 3. Result Bone marrow metastasized cells were exclusively cultured for with the use of high-glucose D-MEM medium and activated FBS which ruled out the growth of precursor cells a n d leukocytes, respectively. After the initial incubation for 24 h, expansion of the spindle-like cells was observed representing that they possess high growth potential (Fig. 1). Cells were trypsinized and passaged when 70% confluence was reached which usually happened every
10-14 days making up a total of 12 passages thus far in a span of 150 days. In our population of patients, cytokeratin was stained positive in more than 90% of the cases in the disseminated cancer cells of bone marrow (Table 1). 4. Discussion We presented here the longest culture system of micrometastasized cells in bone marrow of 150days hitherto. The bone marrow disseminated cancer cells appeared to be anchored onto the culture flask after initial incubation of 24h. Some cultures indicated that micrometastatic cells can inherit a strong growth potential [1]. Culture techniques could increase the sensitivity of detection of occult tumor cells in human bone marrow [ 14]. With our highly selective culture system described in this study, the cancer cells after cell culture would be purer than those isolated b y magnetic-activated cell separation (MACS) (Miltenyi Biotec, Germany) compared with our previous study. The survival of cancer cells from bone marrow would also be lengthened. Cell lines have been established from bone marrow micrometastases transfected with SV40 T-Ag compared to our culture system without involving plasmid construction, as a result their reported culture process could lead to selection of those cell clones that adapt best to the culture conditions [15]. Immunocytochemistry using tumor-associated monoclonal antibodies has led to improvements in the ability to detect occult breast cancer cells in bone marrow aspirates and peripheral blood. Nevertheless, the immunocytochemistry method needs to be further developed before it can be used routinely in the clinic [16]. Analysis of cultured micrometastatic tumor cells could provide a greater number of tumor markers-positive cells available for subsequent molecular analyses. Cytokeratin and is an intermediate filament which present mainly in epithelium. After bone marrow culture, viable epithelial cells detected by CK staining were observed in the bone marrow culture of more than 90% of the patients which is higher than that detected by Solakoglu et al. [17] being 81%. The incidence of CKpositive cultured cancer cells found in our study was also much higher than the percentage of cancer cells stained directly obtained from bone marrow aspirates [ 18]. Recent data show that the presence of occult tumor cells in the bone marrow at diagnosis appears to increase the rate of recurrence in the patients. The results indicate that this Table 1 Antigen characteristics of bone marrowmetastasizedcells in 40 cases
Fig. 1. Histologyof bone marrow metastasizedceils showing spindle cells growing on glass slide. Hematoxylin-eosinstaining. Magnification20x.
$385
Antigen
Bone marrowmetastasizedcells
Cytokeratin
++
Vimentin
++
COX-2
+
HER2/neu
+
$386
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $384-$386
technique can be used as an early diagnostic technique of bone marrow micrometastasis in the patient with breast cancer thereby promoting the development of therapeutic strategy. However, a better understanding of the biology of micrometastatic cells is necessary to increase the precision of detection in bone marrow. Therefore, a long-term culture system of bone marrow metastasized cells is a cornerstone for the mentioned targets to be achieved.
[8]
References
[11]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
Pierga JY, Bonneton C, Magdelnat H, Vincent-Salomon A, Nos C, Pouillart P, Thiery JP. Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer. Br J Cancer 2003;89:539--45. Harbeck N, Untch M, Pache L. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3year median follow-up. Br J Cancer 1994;69:566-71. Diel IJ, Kaufmann M, Costa SD, Holle R, von Minckwitz G, Solomayer EF, Kaul S, Bastert G. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652-8. Mansi JL, Gogas H, Bliss JM, Gazet JC, Berger U, Coombes RC. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999;354:197-202. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G. Cytokeratin-positive cells in the bone marrow and survival of patients with stage 1, !I, or III breast cancer. N Engl J Med 2000b;342:525-33. Gebauer G, Fehm T, Merkle E, Beck EP, Lang N, Jager W. Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001; 19:3669-74. Gerber B, Krause A, Muller H, Richter D, Reimer T, Makowitzky J, Herrnring C, Jeschke U, Kundt G, Friese K. 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;19:960-71.
[9]
[10]
[12]
[13] [14]
[15]
[16]
[ 17
[18]
Pantel K, Dickmanns A, Zippelius A, Klein C, Shi J, HoechtlenVollmar W, Schlimok G, Weckermann D, Obernerder R, Fanning E, et al. Establishment of micrometastatic carcinoma cell lines: a novel source of tumor cell vaccines. J Natl Cancer Inst 1995;87:1162-8. Allgayer H, Heiss MM, Riesenberg R, Grutzner KU, Tarabichi A, Babic R, Schildberg FW. Urokinase plasminogen activator receptor (uPAR): one potential characteristic of metastatic phenotypes in minimal residual tumor disease. Cancer Res 1997;57:1394-9. Muller V, Pantel K. Bone marrow micrometastases and circulating tumor cells: current aspects and future perspectives. Breast Cancer Res 2004;6(6):258-61 [Epub 2004 Sep 29]. Weinschenker P, Snares HP, Clark O, Giglio AD. Immunocytochemical detection of epithelial cells in the bone marrow of primary breast cancer patients: a meta-analysis. Breast Cancer Res Treat 2004 Oct;87(3):215-24. Diel IJ, Solomayer EF, Bastert G. Micrometastatic cells in the bone marrow of patients with breast carcinoma. Radiologe 2000 Aug;40(8):681-7. Braun S, Pantel K. Biological characteristics of micrometastatic cancer cells in bone marrow. Cancer Metastasis Rev 1999;18:75-90. Joshi SS, Novak DJ, Messbarger L, Maitreyan V, Weisenburger DD, Sharp JG. Levels of detection of tumor cells in human bone marrow with or without prior culture. Bone Marrow Transplant 1990;6:179-83. Putz E, Witter K, Offner S, Stosiek P, Zippelius A, Johnson J, Zahn R, Riethmuller G, Pantel K. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases. Cancer Res 1999 Jan 1;59( I ):241-8. Kvalheim G. Detection of occult tumour cells in bone marrow and blood in breast cancer patients--methods and clinical significance. Acta Oncol 1996;35(Suppl 8): 13-8. Solakoglu O, Maierhofer C, Lahr G, Breit E, Scheunemann P, Heumos I, Pichlmeier U, Schlimok G, Oberneder R, Kollermann MW, Kollermann J, Speicher MR, Pentel K. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci USA 2002;99:2246-51. Nakamura T, Matsunami K, Hayashi K, Ota M, Ide H, Takasaki K. Detection of bone marrow micrometastasis in esophageal cancer patients by immunomagnetic separation. Oncol Rep 2004 May; 11 (5):999-1003.
SCIENCE ~DIRECT
ELSEVIER
®
& BIOMEDICINE PHARMACOTHERAPY
Biomedicine & Pharmacotherapy 59 (2005) $384-$386 http://france.elsevier.com/direct/BIOPHA/
The value of bone marrow aspirates culture for the detection of bone marrow micrometastasis in breast cancer W.T.Y. Loo, J.H.M. Fong, L. Zhu, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, Universityof Hong Kong Medical Center, PokfulamRoad, Queen Mary Hospital, Hong Kong, China
Abstract
Background. - Detection of micrometastasis is an important problem of clinical significance for a better understanding and control of tumor progression, which will improve patients' survival time. Tumor cells in bone marrow (BM) aspirates are indicative of the general disseminative metastasis in patients with early breast cancer and characterization of breast cancers by various tumor markers which are appropriate for the identification of high risk groups. M a t e r i a l s and methods. - Bone marrow aspirates were obtained from 44 breast cancer patients at the time of surgery. To identify micrometastases in bone marrow, an immunocytochemical assay for epithelial cytokeratin (CK) was performed at the second passage after selective culture. Cytokeratin-positive bone marrow disseminated cancer cells were observed in more than 90% of the patients. This high incidence needs further investigation with bigger sample size to confirm. However, these results indicate that this technique can be used as an early diagnostic technique of bone marrow micrometastasis in the patient with breast cancer thereby promoting the development of therapeutic strategy. High incidences need further investigation with bigger samples to confirm. © 2005 Elsevier SAS. All rights reserved. Keywords: Bone marrow; Micrometastasis; Cytokeratin; Cyclooxygenase 2
1. Introduction The clinical importance of metastasized cells in the bone marrow of breast cancer patients has been demonstrated in several prospective studies, and represents an independent prognostic factor for distant relapse and overall survival [17]. Very little is known about the biological features of the disseminated cancer cells. However, extensive cell culture experiments have shown that cells disseminating into bone marrow have a time-limited proliferative potential [8]. Immunocytochemical (ICC) detection of epithelial cells in the bone marrow of breast cancer patients has been performed with a variety of antibodies thus far in many similar studies [9-12]. Cytokeratin is intermediate filament which present mainly in epithelium. Currently available data suggest that CK-positive cells in bone marrow aspirates
*
Corresponding author.
E-mail address: [email protected] (L.W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
of cancer patients represent a selected, but nevertheless heterogeneous population of dormant (G0-phase) cancer cells [13]. The aim of the current study was to culture micrometastatic tumor cells present in bone marrow of patients with invasive ductal carcinoma and to thereby establish immortalized cell lines. The established system will allow an in-depth molecular analysis of human micrometastatic cancer cells and could become a useful source for the identification of high risk groups. 2. Material and methods 2.1. Cell culture
Bone marrow aspirates anterior superior iliac spine (ASIS) from 44 patients diagnosed with invasive ductal carcinoma were collected in EDTA tubes given their consent. Once they were transferred to the laboratory, the
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $384-$386
red blood cells of the specimens were lyzed with cell lysis buffer for 5 min before being spun down. The cell pellet containing the tumor cells was resuspended and distributed into a plastic 25 cm 2 cell culture flask (Coming Glass Works, Coming, NY, USA), with high-glucose Dulbecco's modified eagle medium (D-MEM) with 15% fetal bovine serum (FBS), 15 mM HEPES buffer, L-glutamine, pyridoxine hydrochloride, penicillin (100 U/ml), streptomycin (100 ~tg/ml) and insulin (4 ~tg/ml) (Invitrogen Corporation, California, USA). The culture flask was incubated at 37 °C in 95% air and 5% CO 2 for 24 h. All culture medium was removed and replaced with fresh medium every 3 days. 2.2. I m m u n o c y t o c h e m i c a l s t u d i e s
Various antigens of monolayers of cells cultured on 4-well SonicSeal Slide (NUNC, USA) were immunocytochemically tested with mouse monoclonal antibody cytokeratin (Dako Corporation, USA) after five passages. This was performed by using a SuperPicture kit (Zymed Laboratories Inc, USA). Cells were fixed in 4% paraformaldehyde and incubated with primary antibodies in a ratio of 1:100 at 4 °C overnight. Staining was performed using DAB substrate-chromogen solution accordingly. Cells were counterstained with hematoxylin and mounted in Permount. The intensity of the stain was graded as negative (-), weak (+), or strong (++), depending on the number of cells stained and the thickness and darkness of the DAB precipitate. 3. Result Bone marrow metastasized cells were exclusively cultured for with the use of high-glucose D-MEM medium and activated FBS which ruled out the growth of precursor cells a n d leukocytes, respectively. After the initial incubation for 24 h, expansion of the spindle-like cells was observed representing that they possess high growth potential (Fig. 1). Cells were trypsinized and passaged when 70% confluence was reached which usually happened every
10-14 days making up a total of 12 passages thus far in a span of 150 days. In our population of patients, cytokeratin was stained positive in more than 90% of the cases in the disseminated cancer cells of bone marrow (Table 1). 4. Discussion We presented here the longest culture system of micrometastasized cells in bone marrow of 150days hitherto. The bone marrow disseminated cancer cells appeared to be anchored onto the culture flask after initial incubation of 24h. Some cultures indicated that micrometastatic cells can inherit a strong growth potential [1]. Culture techniques could increase the sensitivity of detection of occult tumor cells in human bone marrow [ 14]. With our highly selective culture system described in this study, the cancer cells after cell culture would be purer than those isolated b y magnetic-activated cell separation (MACS) (Miltenyi Biotec, Germany) compared with our previous study. The survival of cancer cells from bone marrow would also be lengthened. Cell lines have been established from bone marrow micrometastases transfected with SV40 T-Ag compared to our culture system without involving plasmid construction, as a result their reported culture process could lead to selection of those cell clones that adapt best to the culture conditions [15]. Immunocytochemistry using tumor-associated monoclonal antibodies has led to improvements in the ability to detect occult breast cancer cells in bone marrow aspirates and peripheral blood. Nevertheless, the immunocytochemistry method needs to be further developed before it can be used routinely in the clinic [16]. Analysis of cultured micrometastatic tumor cells could provide a greater number of tumor markers-positive cells available for subsequent molecular analyses. Cytokeratin and is an intermediate filament which present mainly in epithelium. After bone marrow culture, viable epithelial cells detected by CK staining were observed in the bone marrow culture of more than 90% of the patients which is higher than that detected by Solakoglu et al. [17] being 81%. The incidence of CKpositive cultured cancer cells found in our study was also much higher than the percentage of cancer cells stained directly obtained from bone marrow aspirates [ 18]. Recent data show that the presence of occult tumor cells in the bone marrow at diagnosis appears to increase the rate of recurrence in the patients. The results indicate that this Table 1 Antigen characteristics of bone marrowmetastasizedcells in 40 cases
Fig. 1. Histologyof bone marrow metastasizedceils showing spindle cells growing on glass slide. Hematoxylin-eosinstaining. Magnification20x.
$385
Antigen
Bone marrowmetastasizedcells
Cytokeratin
++
Vimentin
++
COX-2
+
HER2/neu
+
$386
W.T.Y. Loo et al. / Biomedicine & Pharmacotherapy 59 (2005) $384-$386
technique can be used as an early diagnostic technique of bone marrow micrometastasis in the patient with breast cancer thereby promoting the development of therapeutic strategy. However, a better understanding of the biology of micrometastatic cells is necessary to increase the precision of detection in bone marrow. Therefore, a long-term culture system of bone marrow metastasized cells is a cornerstone for the mentioned targets to be achieved.
[8]
References
[11]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
Pierga JY, Bonneton C, Magdelnat H, Vincent-Salomon A, Nos C, Pouillart P, Thiery JP. Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer. Br J Cancer 2003;89:539--45. Harbeck N, Untch M, Pache L. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3year median follow-up. Br J Cancer 1994;69:566-71. Diel IJ, Kaufmann M, Costa SD, Holle R, von Minckwitz G, Solomayer EF, Kaul S, Bastert G. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652-8. Mansi JL, Gogas H, Bliss JM, Gazet JC, Berger U, Coombes RC. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999;354:197-202. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G. Cytokeratin-positive cells in the bone marrow and survival of patients with stage 1, !I, or III breast cancer. N Engl J Med 2000b;342:525-33. Gebauer G, Fehm T, Merkle E, Beck EP, Lang N, Jager W. Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001; 19:3669-74. Gerber B, Krause A, Muller H, Richter D, Reimer T, Makowitzky J, Herrnring C, Jeschke U, Kundt G, Friese K. 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;19:960-71.
[9]
[10]
[12]
[13] [14]
[15]
[16]
[ 17
[18]
Pantel K, Dickmanns A, Zippelius A, Klein C, Shi J, HoechtlenVollmar W, Schlimok G, Weckermann D, Obernerder R, Fanning E, et al. Establishment of micrometastatic carcinoma cell lines: a novel source of tumor cell vaccines. J Natl Cancer Inst 1995;87:1162-8. Allgayer H, Heiss MM, Riesenberg R, Grutzner KU, Tarabichi A, Babic R, Schildberg FW. Urokinase plasminogen activator receptor (uPAR): one potential characteristic of metastatic phenotypes in minimal residual tumor disease. Cancer Res 1997;57:1394-9. Muller V, Pantel K. Bone marrow micrometastases and circulating tumor cells: current aspects and future perspectives. Breast Cancer Res 2004;6(6):258-61 [Epub 2004 Sep 29]. Weinschenker P, Snares HP, Clark O, Giglio AD. Immunocytochemical detection of epithelial cells in the bone marrow of primary breast cancer patients: a meta-analysis. Breast Cancer Res Treat 2004 Oct;87(3):215-24. Diel IJ, Solomayer EF, Bastert G. Micrometastatic cells in the bone marrow of patients with breast carcinoma. Radiologe 2000 Aug;40(8):681-7. Braun S, Pantel K. Biological characteristics of micrometastatic cancer cells in bone marrow. Cancer Metastasis Rev 1999;18:75-90. Joshi SS, Novak DJ, Messbarger L, Maitreyan V, Weisenburger DD, Sharp JG. Levels of detection of tumor cells in human bone marrow with or without prior culture. Bone Marrow Transplant 1990;6:179-83. Putz E, Witter K, Offner S, Stosiek P, Zippelius A, Johnson J, Zahn R, Riethmuller G, Pantel K. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases. Cancer Res 1999 Jan 1;59( I ):241-8. Kvalheim G. Detection of occult tumour cells in bone marrow and blood in breast cancer patients--methods and clinical significance. Acta Oncol 1996;35(Suppl 8): 13-8. Solakoglu O, Maierhofer C, Lahr G, Breit E, Scheunemann P, Heumos I, Pichlmeier U, Schlimok G, Oberneder R, Kollermann MW, Kollermann J, Speicher MR, Pentel K. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci USA 2002;99:2246-51. Nakamura T, Matsunami K, Hayashi K, Ota M, Ide H, Takasaki K. Detection of bone marrow micrometastasis in esophageal cancer patients by immunomagnetic separation. Oncol Rep 2004 May; 11 (5):999-1003.