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Japanese Journal of Clinical Oncology Pages 480-485

Immunohistochemical Detection of Tumor Cells in the Bone Marrow of Breast Cancer Patients
Introduction
Materials And Methods
   Breast Cancer Cell Lines
   Monoclonal Antibodies
   Bone Marrow Cells
   Immunohistochemical Staining Method
   Flow Cytometry
Results
   Reactivity of Breast Cancer Cell Lines with the MoAbs (Table 1)
   Reactivity of Normal Bone Marrow Cells with MoAbs Determined by Immunohistochemistry (Table 3)
   Detection of Contaminant Cells
   Immunohistochemical Detection of Contaminant Tumor Cells in the Bone Marrow of Patients with Advanced or Metastatic Breast Cancer
Discussion
Acknowledgments
References
Immunohistochemical Detection of Tumor Cells in the Bone Marrow of Breast Cancer Patients

Immunohistochemical Detection of Tumor Cells in the Bone Marrow of Breast Cancer Patients

Akira Okumura, Yutaka Tokuda, Makiko Tanaka, Hiroyasu Makuuchi and Tomoo Tajima

Department of Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Background: Contamination of bone marrow and peripheral blood stem cells with tumor cells is a problem that may be encountered when autologous hematopoietic stem cell transplantation is conducted concurrently with high-dose chemotherapy.
Methods: Using monoclonal antibodies to a variety of tumors, the detection of tumor cells in the bone marrow of breast cancer patients was studied by immunohistochemistry.
Results: KL-1 and CAM5.2 were strongly reactive with breast cancer cells, but not with normal bone marrow cells. The reactivity of the tumor cells with EMA was not strong, and DF-3 and 115D8 yielded only slightly positive reactions. These latter antibodies also exhibited some reactivity to normal bone marrow cells. When tumor cells were admixed with normal cells, the sensitivity of CAM5.2 and EMA permitted the detection of one cell in 104, but with KL-1, the detection of one in 105 cells was possible. When immunohistochemical staining was used in testing 40 patients with advanced or recurrent breast cancer, positive reactions were obtained in four of 27 patients (14.8%) with KL-1, four of 26 (15.4%) with CAM5.2, and nine of 37 (23.7%) with KL-1 + CAM5.2, figures similar to those reported by others who studied stage IV patients.
Conclusions: Immunohistochemical staining with KL-1 and CAM5.2 is therefore considered to be a useful technique for detecting contamination by tumor cells.

Key words: tumor detection - keratin - KL-1 - CAM5.2 - immunohistochemistry

INTRODUCTION

The combination of autologous hematopoietic stem cell transplantation and high-dose chemotherapy is a promising therapeutic technique in advanced cases of breast cancer. In our institution, we have used autologous bone marrow and peripheral blood stem cell transplantation with high-dose chemotherapy since 1980 and 1991, respectively, both for advanced cases of recurrent breast cancer and postoperative multiple lymph node metastases (1). Contamination of the autologous hematopoietic stem cells with breast cancer cells has been a problem in these procedures. Moreover, the presence of contaminant breast cancer cells in the bone marrow has been reported to serve as a predictive factor in postoperative cases (2). The detection of contaminant breast cancer cells has been the subject of many studies, but conventional histochemical tests have proven unsatisfactory for this purpose. There have also been many reports on immunohistochemical staining methods using monoclonal antibodies (MoAbs), but none addressed the problem from the viewpoint of basic medicine. We therefore undertook basic research on many questions concerning the reactivity of MoAbs and sought to identify which antibodies were effective in detecting contamination by breast cancer cells.

MATERIALS AND METHODS

Breast Cancer Cell Lines

The breast cancer cell lines SK-BR-3, MDA-MB-453, ZR75-1 and MCF7 were purchased from the American Type Culture Collection (Rockville, MD, USA). The cell lines were cultured in either RPMI 1640 or Dulbecco's Modified Eagle Medium (Hazelton Research Products, Kansas City, MO, USA), containing 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY, USA). All the cell lines tested negative for mycoplasma contamination. Before use, the cell lines were processed in 0.25% trypsin and 0.15% ethylenediaminetetraacetic acid (EDTA) in an incubator at 37°C, then carefully scraped free from the wall of the incubating dishes. The cells were then washed in RPMI 1640 containing 10% FBS, and a viable cell count was made.

Monoclonal Antibodies

KL-1 (Immunotech, Marseille Cedex, France) (3), CAM5.2 (Becton-Dickinson, Mountain View, CA, USA) (4), EMA (EMA-E29) (DAKO, Glostrup, Denmark) (5), 115D8 (Toray-Fuji-Bionics, Tokyo, Japan) (6) and DF-3 (Japan Tanner, Tokyo, Japan) (7) were used. KL-1 is a MoAb to keratin obtained from the keratinous layer of normal human skin, and recognizes keratins 1, 2, 3, 4, 10 and 11. CAM5.2 is a MoAb to keratin obtained from colon cancer cell line HT29, and recognizes keratins 8, 18 and 19. EMA is a MoAb against the epithelial cell membrane, and the antigens that it recognizes belong to the human milk-fat globule heterogeneous group of heavily glycosylated proteins. 115D8 is a MoAb against the highly purified human milk-fat globule membrane. Finally, DF-3 is a MoAb to the cell membrane of metastatic breast cancer cells and reacts with 290 kDa glycoproteins.

Bone Marrow Cells

Normal bone marrow cells were harvested from the posterior iliac bone of donors for allogenic bone marrow transplantation. Other bone marrow cells were collected from the posterior iliac bone of 40 patients with advanced or recurrent breast cancer. Isolation was by the method of Gilmore et al. (8), using a Cobe 2991 Blood Cell Processor (Cobe Laboratories, Lakewood, CO, USA). The bone marrow cells (1 × 107/ml) were centrifuged for 15 min at 800 r.p.m., fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature, then again centrifuged at 800 r.p.m. for 15 min in a cytospin process that yielded 1.5 × 106 cells per slide. This was followed by additional fixation in 4% PFA for 40 min. The slides were then washed for 5 min with each of the following: 5% sucrose, 10% sucrose, 0.01 M phosphate-buffered saline (PBS) and 5% normal rabbit serum (NRS).

Immunohistochemical Staining Method

The breast cancer cell lines (1 × 106) were subjected to the cytospin process, then prepared as were the bone marrow cells and affixed. The MoAbs KL-1, CAM5.2, EMA, 115D8 and DF-3, 1 µg of protein/ml each, were added to the various normal and experimental cell slide preparations and reacted for 30 min at 4°C, following which they were washed three times with 0.5% bovine serum albumin (BSA/PBS). The slides were pre-treated with levamisole (Sigma, St Louis, MO, USA) to inhibit the activation of endogenous alkaline phosphatase. The reactions of the cells with the antibodies were determined with alkaline phosphatase-conjugated rabbit anti-mouse IgG (DAKO, Glostrup, Denmark). The reactive cells stained a strong violet color, and were easily distinguished from non-stained cells. The diagnostic criteria were based only on reactivity, not on any morphology.

Table 1. Immunohistochemical reactivity of MoAbs to breast cancer cells
  KL-1 CAM5.2 Cocktail EMA DF3 115D8
SK-BR-3 97.0* 95.3 97.3 23.7 29.8 49.0
ZR75-1 97.1 94.2 97.5 89.6 90.6 96.9
MCF7 NT[dagger] NT 98.2 15.4 NT NT
MDA-MB-453 NT NT 98.6 NT NT NT
*% positive/1000 cells. NT, Not tested.

Table 2. Flow cytometric reactivity of MoAbs to breast cancer cells
Cells % of cells positive with MoAbs
KL-1 CAM5.2 KL-1 + CAM5.2 EMA
SK-BR-3 95.0 93.5 95.2 86.5
MDA-MB-453 95.4 91.2 95.5 25.0
MCF-7 98.5 96.6 99.1 39.3
ZR-75-1 87.6 85.3 87.6 97.7
Total* 94.1 ± 4.6 91.7 ± 4.8 94.4 ± 4.8 62.1 ± 39.4
*All values are expressed as the mean ± SD.

Flow Cytometry

Breast cancer cells were centrifuged for 5 min at 1200 r.p.m., then washed. After adding 5% BSA, the cells were washed again for 5 min at 1200 r.p.m. Next, the cells were fixed for 30 min with 4% PFA at 4°C, washed twice with 0.5% BSA/PBS, and resuspended in this solution at a concentration of 1 × 106/100 µl. Forty microliters of KL-1, CAM5.2 or EMA were added to a final concentration of 1 µg of protein/ml, and the resulting mixture incubated at 4°C for 30 min. Next, the cells were washed three times with 0.5% BSA/PBS solution. Following this, the secondary antibody, fluorescein-conjugated goat anti-mouse IgG (TAGO, Burlingame, CA, USA), was added at a concentration of 10:1 in terms of the amounts of protein, and the reaction allowed to proceed for 20 min at 4°C. Finally, after being washed twice with 5% BSA/PBS, the cells were sorted with a fluorescence-activated cell sorter (FACStar, Becton-Dickinson, Mountain View, CA, USA).

RESULTS

Reactivity of Breast Cancer Cell Lines with the MoAbs (Table 1)

With the above immunohistochemical staining technique, most of the SK-BR-3 cells gave a strong positive staining reaction with MoAbs KL-1 and CAM5.2 (97 and 95.3%, respectively), while with EMA, DF-3 and 115D8, fewer cells stained (23.7, 29.8 and 49%, respectively), and the reactions were not as strong (Fig. 1). In addition, when the positive cell ratio was defined as the number of cells with a greater intensity than the controls and expressed as a proportion of the total, flow cytometry yielded the following results: KL-1, 94.1 ± 4.6; CAM5.2, 91.7 ± 4.8; both KL-1 and CAM5.2, 94.4 ± 4.8; and EMA, 62.1 ± 39.4 (means ± SD). The overall ratio with EMA was low, but this appears to be because the values for MDA-MB-453 and MCF-7 were unusually low (Table 2).


Figure 1. Reactivity of SK-BR-3 cells to MoAbs. (a) KL-1 + CAM5.2: strong positive reaction (×40). (b) Anti-EMA: intermediate reaction (×40). (c) 115D8: weak reaction (×40). (d) DF3: weak reaction (×40).

Table 3. Reactivity of MoAbs to normal bone marrow mononuclear cells
MoAb Reactivity*
KL-1 0
CAM5.2 0
EMA 24
DF-3 14
115D8 15
*Number positive/1000 cells.

Table 4. Detection of contaminant breast cancer cells among bone marrow mononuclear cells
Tumor cells:bone marrow cells KL-1 CAM5.2 EMA
1:1000 + + +
1:10 000 + + +
1:100 000 + - -

Reactivity of Normal Bone Marrow Cells with MoAbs Determined by Immunohistochemistry (Table 3)

Normal bone marrow cells were reactive with EMA, DF-3 and 115D8 (Fig. 2), but not with KL-1 and CAM5.2.

Detection of Contaminant Cells

With the use of CAM5.2 and EMA, a single tumor cell (SK-BR-3 cell) could be detected among 104 normal bone marrow cells, but with KL-1, the ratio increased to one in 105 cells [Table 4, Fig. 3(a) and (b)].


Figure 2. False-positive reactivity of normal bone marrow cells to MoAbs. Some cells show staining using DF3.


Figure 3. Artificial contaminant SK-BR-3 detection in normal bone marrow. Cells and tumor cell detection in bone marrow of breast cancer patient. (a) Tumor:normal = 1:105 (anti-EMA)-negative. (b) Tumor:normal = 1:105 (KL-1)-positive. (c) Positive staining of contaminant tumor cell in bone marrow of patient no. 32 using KL-1 plus CAM5.2 (cocktail).

Immunohistochemical Detection of Contaminant Tumor Cells in the Bone Marrow of Patients with Advanced or Metastatic Breast Cancer

We also collected data on the detection of contaminant tumor cells in the bone marrow of patients with advanced or metastatic cancer by an immunohistochemical method using KL-1, CAM5.2 and EMA. The results are presented in Table 5 and Fig. 3(c). When KL-1 was used, 4/27 tests (14.8%) were positive; with CAM5.2, the ratio was 4/26 (15.4%); with KL-1 + CAM5.2, it was 9/37 (23.7%); and with EMA, 9/17 (52.9%). In 13 cases in which the same specimen was examined with a combination of KL-1 and CAM5.2, and with EMA, there were two (15.4%) positive results with KL-1 + CAM5.2 and seven (53.8%) with EMA.

DISCUSSION

During autologous hematopoietic stem cell transplantation, contamination with tumor cells may occur. However, no technique has as yet been established for identifying such contaminant tumor cells, although a wide variety of methodologies have been examined. The methods of analysis have included flow cytometry, immunohistochemical staining and cytological diagnosis. For flow cytometry and immunohistochemistry, MoAbs against breast cancer, epithelial cells and cytokeratin were used. In the past, attempts to detect contaminant cells by cytology involved procedures of extremely low sensitivity and therefore of little use (9). The sensitivity of flow cytometry seems to vary with the investigator, with the highest level of sensitivity reported thus far being one in 104. However, with immunohistochemistry, a sensitivity of one in 105 has been reached. Leslie et al. (10) considered that a detection sensitivity of one in 104 was possible in flow cytometry if the mean intensity was compared with the control. Molino et al. (9) reported that whereas the maximum sensitivity obtainable with flow cytometry was one in 5 × 102, immunohistochemical staining could offer detection at a level of one in 4 × 105. These reports indicate that immunohistochemical staining is the best technique for detecting contaminant tumor cells. Accordingly, we sought to ascertain which would be the best MoAb to use. The MoAbs we studied were DF-3 and 115D8 against breast cancer; EMA, a MoAb frequently used in both Europe and the USA; and, bearing in mind the fact that bone marrow cells have no components derived from epithelium, KL-1 and CAM5.2, which are MoAbs against keratin.

When the breast cancer cell line SK-BR-3 was used in investigating the reactivity of tumor cells with these MoAbs by examining the ratio of positively stained cells and the degree of staining, it was found that EMA gave the lowest positive ratio, DF-3 and 115D8 yielded higher ratios, and KL-1 and CAM5.2 provided the highest positive ratios. As to the strength or weakness of staining, KL-1 and CAM5.2 gave very good results. When KL-1 and CAM5.2 were compared with EMA by flow cytometry, it was seen that EMA had a ratio of positive staining that was much lower than those of the preceding two. Moreover, when a cocktail of KL-1 and CAM5.2 was used, the ratio increased, though only to a small extent.

Next, the reactivity of these MoAbs with normal bone marrow cells was examined. DF-3, 115D8 and EMA were reactive with these cells, whereas no such reaction was obtained with KL-1 and CAM5.2. The reactivity of EMA with cells other than tumor cells was reported by Ellis et al. (12) investigating non-epithelial cells and lymphocytes, and by Thor et al. (11) studying blood plasma and immature cells. Thor et al. (11) also pointed out that the reactivity of DF-3 was similar to that of EMA, but that CAM5.2 showed no reactivity with normal bone marrow cells.

As the above indicates, KL-1 and CAM5.2 are highly reactive with tumor cells, but not with normal bone marrow cells. They are, therefore, suitable as reagents for the detection of contaminant tumor cells.

Study of the sensitivity of KL-1, CAM5.2 and EMA in detecting contaminant tumor cells showed a ratio of one in 104 for CAM5.2 and EMA, while with KL-1 it was 1 in 105. The sensitivity of the method using KL-1 is on a par with the highest levels of sensitivity previously reported for the detection of tumor cells with immunohistochemical staining. Mathieu et al. (13) made experimental comparisons between KL-1, AE1-AE3, CAM5.2, EMA and HMFG-2, and reported that KL-1 was the most sensitive. The studies described above clearly indicate that for the detection of tumor cells as contaminants in bone marrow, KL-1 possesses the highest specificity and the greatest accuracy. However, the action of KL-1 is related to keratins 1, 2, 3, 4, 10 and 11, whereas that of CAM5.2 is to keratins 8, 18 and 19. It was considered that more effective epithelial cell detection would result from combining antibodies to as many types of keratin as possible.

It was reported recently that keratin 19 (CK-19) is not present in either normal lymph nodes or normal bone marrow cells, and that lymph node metastases can be detected with a high degree of sensitivity (one cell in 106) by means of a reverse transcriptase-polymerase chain reaction (RT-PCR) using CK-19 (14). In the study of Seiden et al. (15), however, when they employed the RT-PCR using CK-19 to detect tumor cells in normal human peripheral blood, no positives were seen after 50 cycles, but after 70 cycles eight of 14 cases (57%) were positive. This technique can hardly be said to be a practical method of detection. In addition, although the RT-PCR can offer a qualitative diagnosis, quantitatively it is far from satisfactory, and so the usefulness of immunohistochemical staining as a method of detection is greater.

Table 5. Immunohistochemical staining of bone marrow cells from patients with primary or recurrent breast cancer
Case TNM stage KL-1 CAM5.2 Cocktail* Anti-EMA
1 T1N1M0 - - - NT
2 recurrent - - - NT
3 T4bN3M0 - - - NT
4 T1N2M0 - - - -
5 recurrent - + - +
6 T1N1M0 - + + NT
7 T3N0M0 - + + NT
8 T3N2M0 - - - -
9 T4cN0M0 - - - NT
10 T2N2M1 + - + +
11 T2N1M0 NT NT - NT
12 T2N2M0 NT NT + NT
13 recurrent - - - -
14 T3N2M0 - - - +
15 recurrent - - - -
16 T3N1M0 - - - NT
17 recurrent - - - NT
18 T1N1M0 - - - +
19 T3N3M0 - - - NT
20 T4cN3M1 NT NT + NT
21 recurrent - - NT -
22 T2N2M0 NT NT - NT
23 T3N2M0 - - - +
24 T2N1M0 NT NT - NT
25 recurrent + - NT +
26 T2N2M0 NT NT - NT
27 recurrent NT NT + NT
28 recurrent + NT NT -
29 T3N2M0 NT NT + NT
30 T1N0M0 NT NT - NT
31 recurrent + + + +
32 T3N2M0 NT NT + NT
33 T2N1M0 - - - NT
34 recurrent NT NT - NT
35 recurrent - - - NT
36 recurrent - - - -
37 recurrent NT NT - NT
38 recurrent - - - -
39 recurrent NT NT - NT
40 recurrent - - - +
*Cocktail: KL-1 plus CAM5.2. NT, Not tested.

In our institution, we compared two variations of the immunohistochemical staining method for tumor cell detection within the bone marrow of advanced recurrent breast cancer patients of a type generally treated with massive-dose chemotherapy. One variation tested both KL-1 and CAM5.2, and the other tested EMA, which has been frequently studied in the USA and Europe. The detection of contaminant tumor cells in bone marrow is mainly used as a predictive factor (16), but there are few reports that focus specifically on advanced recurrent cancer patients, and none that make direct comparisons of anti-keratin antibodies with EMA. However, Mansi et al. (17), using EMA, successfully detected contaminant cells in the bone marrow of 13 of 20 cases (65%) of recurrent breast cancer. Also, Ross et al. (18) reported 28 positives (70%) among 40 cases of stage IV carcinoma with the use of MoAbs to breast or glandular epithelia. In contrast, Singletary et al. (19), using three MoAbs to keratin (AE1, AE3 and MAK-6), found positive reactions in only 38% of 71 stage IV cases. The results of our study are similar, showing a 23.7% rate of positives when both KL-1 and CAM5.2 were used, as opposed to a rate of 52.9% with the use of EMA alone. The MoAb against keratin gave a lower ratio of positives than EMA, but this was thought to be due to the number of false positives generated by the use of EMA for immunohistochemical staining.

Acknowledgments

This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan. The authors wish to thank Mr C. W. P. Reynolds for the preparation of this manuscript.

References

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2. Landys K. Prognostic value of bone marrow biopsy in breast cancer. Cancer 1982;49:513-8. MEDLINE Abstract

3. Viac J, Reano A, Brochier J, Staquet MJ, Thivolet J. Reactivity pattern of a monoclonal antikeratin antibody (KL-1). J Invest Dermatol 1983;81:351-4. MEDLINE Abstract

4. Makin C, Bobrow L, Bodmer W. Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 1984;37:975-83. MEDLINE Abstract

5. Heyderman E, Strudley I, Powell G, Richardson TC, Cordell JL, Mason DY. A new monoclonal antibody to epithelial membrane antigen (EMA)-E29. A comparison of its immunocytochemical reactivity with polyclonal anti-EMA antibodies and with another monoclonal antibody, HMFG-2. Br J Cancer 1985;52:355-61. MEDLINE Abstract

6. Hilkens J, Buijs F, Hilgers J, Hageman P, Calafat J, Sonnenberg A, et al. Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer 1984;34:197-206. MEDLINE Abstract

7. Kufe D, Inghirami G, Abe M, Hayes D, Justi Wheeler H, Schlom J. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma 1984;3:223-32. MEDLINE Abstract

8. Gilmore MJ, Prentice HG, Blacklock HA, Janossy G, Hoffbrand AV. A technique for rapid isolation of bone marrow mononuclear cells using Ficoll-Metrizoate and the IBM 2991 blood cell processor. Br J Haematol 1982;50:619-26. MEDLINE Abstract

9. Molino A, Colombatti M, Bonetti F, Zardini M, Pasini F, Perini A, et al. A comparative analysis of three different techniques for the detection of breast cancer cells in bone marrow. Cancer 1991;67:1033-6. MEDLINE Abstract

10. Leslie DS, Johnston WW, Daly L, Ring DB, Shpall EJ, Peters WP, et al. Detection of breast carcinoma cells in human bone marrow using fluorescence-activating cell sorting and conventional cytology. Am J Clin Pathol 1990;94:8-13. MEDLINE Abstract

11. Thor A, Viglione MJ, Ohuchi N, Simpson J, Steis R, Cousar J, et al. Comparison of monoclonal antibodies for the detection of occult breast carcinoma metastases in bone marrow. Breast Cancer Res Treat 1988;11:133-45. MEDLINE Abstract

12. Ellis G, Ferguson M, Yamanaka E, Livingston RB, Gown AM. Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients. Cancer 1989;63:2509-14. MEDLINE Abstract

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14. Datta YH, Adams PT, Drobyski WR, Either SP, Terry VH, Roth MS. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994;12:475-82. MEDLINE Abstract

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Received January 5, 1998; accepted May 8, 1998
For reprints and all correspondence: Yutaka Tokuda, Department of Surgery, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan. E-mail: tokuda{at}is.icc.u-tokai.ac.jp
Abbreviations: MoAbs, monoclonal antibodies; FBS, fetal bovine serum; EDTA, ethylenediaminetetraacetic acid; PFA, paraformaldehyde; PBS, phosphate-buffered saline; NRS, normal rabbit serum; BSA, bovine rabbit albumin; CK-19, keratin-19; RT-PCR, reverse transcriptase-polymerase chain reaction

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