ter chemotherapy with VP16, ifosfamide, and cisplatin. (VIP) followed by granulocyte colony-stimulating factor. ERIPHERAL BLOOD progenitor cells (PBPC) are ...
RAPID COMMUNICATION
Mobilization of Tumor Cells and Hematopoietic Progenitor Cells Into Peripheral Blood of Patients With Solid Tumors By Wolfram Brugger, Klaus J. Bross, Maria Glatt, Frauke Weber, Roland Mertelsmann, and Lothar Kanz Peripheral blood progenitor cells (PBPCs) are increasingly used for autografting after high-dose chemotherapy. One advantage of PBPCs over the use of autologous bone marrow wouldbe a reduced risk of tumor-cellcontamination. However, the actual level of tumor cells contaminating PBPC harvests is poorly investigated. It is currently not known whether mobilization of PBPCs might also result in mobilization of tumor cells. We evaluated 358 peripheral blood samples from 46 patients with stage IV or high-risk stage 11/111 breast cancer, small cell (SCLC) or non-small cell (NSCLC) lung cancer, as well as other advanced malignancies for the detection of epithelial tumor cells. Monoclonal antibodies against acidic and basic cytokeratin components and epithelial antigens (HEA) were used in an alkaline phosphatase-anti-alkaline phosphatase assay with a sensitivity of 1 tumor cell within 4 X 1Os total cells. Before initiation of PBPC mobilization, circulating tumor cells were detected in 2/7 (29%)patients with stage IV breast cancer and in 2/10 (20%)patients with extensivedisease SCLC, respectively. In these patients, an even higher number of circulating tumor cells was detected after chemotherapy with VP16, ifosfamide, and cisplatin (VIP) followed by granulocyte colony-stimulating factor
(G-CSF). This approach has previously been shown t o be highly effective in mobilizing PBPCs. In the 42 patients without circulating tumor cells during steady state, tumor cells were mobilized in 9/42 (21%) patients afterVIP GCSF induced recruitment of PBPCs. The overall incidence of tumor cells varied between 4 and 5,600 per 1.6 X 1OB mononuclear cells analyzed. All stage IV breast cancer patients and 50% of SCLC patients were found to concomitantly mobilize tumor cells and PBPCs. Kinetic analyses showed t w o patterns of tumor cell recruitmentdepending on the presence or absence of bone marrow disease: (1) early after chemotherapy (between days 1 and 7 ) in patients withoutmarrow infiltration, and (2) between days 9 and 16 in patients with marrow infiltration, ie, within the optimal time period for the collection of PBPCs. We show that there is a high proportion of patients with circulating tumor cells under steady-state conditions, and in addition a substantial risk of concomitant tumor cell recruitment upon mobilization of PBPCs, particularly in stage IV breast cancer patients with bone marrow infiltration. The biologic and clinical significance of this finding is unknown at present. 0 1994 by The American Society of Hematology.
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preceding the harvest of PBPCs in terms of reduced tumor cell contamination. Although there are reports demonstrating thepresence of circulating tumor cells in children with disseminated neurobla~toma,'~.'' in patientswith l y m p h ~ m a , ' ~or , ' ' in patients with metastatic breast cancer,'* the actual risk oftumor cell contamination in preparations of mobilized PBPCs is not known. Therefore, we investigated whether tumor cells might be recruited concomitant with hematopoietic progenitor cells upon chemotherapy plus granulocyte CSF (GCSF)-induced mobilization of PBPCs. We applied a highly sensitive immunocytochemical assay for the detection of circulating tumor cells in patientswith a variety of malignancies eligible for high-dose chemotherapy and PBPC transplantation. A total of 358 blood samples from 46 cancer patients were evaluated after mobilization of PBPCs by conventional-dose chemotherapy with VP16, ifosfamide, and cisplatin plus G-CSF administration.6 We show that there is a substantial risk of tumor cell mobilization upon recruitmentof PBPCs.
ERIPHERAL BLOOD progenitor cells (PBPC) are increasingly used as an alternative to autologous bone marrow (BM) rescue for patients with solid tumors as well as hematologic malignancies."5 Advantages of this method over the use of autologous marrowinclude the ability to collect PBPCs without general anesthesia in an outpatientsetting, the possibility to harvest progenitor cells in patients with BM fibrosis resulting from pelvic irradiation, and particularly, a more rapid hematopoietic r e ~ o v e r y . ~ PBPCs .~ can be harvested during therecovery phase after myelosuppressive ~hemotherapy,~ afteradministration of colonystimulating factors (CSFS),',~or after chemotherapy plus CSFadrnini~tration.~~'~-'* At our institution, PBPCs are mobilized by conventional dose chemotherapy CSFs to combine a treatment regimen with broad antitumor activity with the simultaneous mobilization of PBPCS.~.".'~ Moreover, chemotherapy CSF induced mobilization results in higher numbers of progenitor cells'0*''and there might be a possible advantage of a therapeutic chemotherapyregimen
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From the Department of Hematology/Oncology. Albert-Ludwigs University Medical Center, Freiburg, Germany. Submitted August23, 1993;accepted October 23, 1993. Address reprint requests to Lothar Kanz, MD,University of Freiburg Medical Center, Department of Hematology/Oncology, Hugstetter Str 55, 79106 Freiburg, Germany. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section I734 solely to indicate thisfact. 0I994 by TheAmerican Societyof Hematology. 0006-4971/94/8303-0037$3.00/0 636
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PATIENTS AND METHODS
Patients. Forty-six consecutive patients with histologically confirmed solid tumors were analyzed. The study protocol was a p proved by the institutional review board and all patients gave written informed consent.The following patients were included newly diagnosed extensive or limited disease small cell (SCLC, n = 17) or non-small cell (NSCLC, n = 12) lung cancer,stage IV breast cancer (n = 7), stage II/III (n = 2) breast cancer patientswith more than 10 positive lymph nodes, as well as any otheradvanced solid tumor for which no standard treatment was available (n = 8; poorly differentiated carcinoma of unknown primary siteand advanced head and neck cancer) (Table I). Blood, Vol83, No 3 (February 1 ), 1994:pp 636-640
637
MOBILIZATION OF TUMOR CELLS AND BLOOD STEM CELLS Table 1. Patient Characteristics and Status of Circulatina Tumor Cells Total No. of Patients With CK/ HEA-posltive Blood Samples Total (n = 46)
Sex (female/male) Median age (range) No. of patients with BM infiltration Tumor localization LD-SCLC ED-SCLC ED-NSCLC Stage IV breast Stage 11/111 breast Miscellaneous
Steady State
After PBPC Recruitment
20/26 47 (23-58)
munostaining assay contained a negative as well as a positive control. One spot with an IgG 1 isotype servedas a negative control, one spot with a human breast cancer cell line (MCF7; kindly provided by H.H. Fiebig, Freiburg,Germany) served as a positive control. A total of 4 spots with at least 1.6 X I O6 total cells were analyzed for the presence of tumor cells using a standard microscope. The total number of cells present on each spot was calculated by counting the cell number within I8 quadrants out of 8 I total quadrants using a special grid withinthe ocular. Based on a statistical model,the error in the calculated cell number was lessthan 6%.
8/46 ( 1 7.4%) 7 10 12
0
0
2
5 1
RESULTS
The APAAP assay was performed with anticytokeratin antibodies (AEI/AE3, KLI) and anepithelial cell antibody 7 2 7 (HEA 125) and is used diagnostically in our departmentfor 0 0 2 the detection of epithelial tumor cells in bothBM specimen, 8 0 0 as well as pleural and peritoneal effusions. In addition to All tumors 46 4 13 anticytokeratin antibodies, we also evaluated antichromogranin, antisynaptophysin, and antineuron specific enolase Abbreviation: CK, cytokeratin. (NSE) antibodiesthat, however, did not show reproducible results in our laboratory. Therefore, AEI/AE3, KLI, and Standard-dose VIP chemotherapy + G-CSFfor the mobilization HEA 125 were the antibodies used in this study because of of PBPCs. Patients were treated with standard-dose VP16 (500 their strong stainingwith epithelial tumor cells. Normal pemg/m2), ifosfamide (4g/m2), and cisplatin (50 mg/m2) (VIP regiripheral blood MNCs (as studied in 18 volunteers) as well men) followed by the administration of G-CSF(Filgrastim,Neupoas BM and peripheral blood specimens from patients with gen; AMGEN, Munich, Germany; 5 pglkg SC starting day 1 after hematologic disorders (asstudied in 32 patients) were nonchemotherapy for 14 days), as described.6,’2 Serial samples ofpereactive with anticytokeratin and HEA 125 antibodies. In ripheral blood(n = 358) were analyzedafter the first and/or second some cases plasma cells were stained weakly by HEA- 125; cycle ofchemotherapy. Each patient was monitored before chemothese cells could be easily discriminated from tumor cells by therapy (steady state)and subsequently three times a week for the presence of tumor cells in peripheral blood samples. At least six morphologic criteria. blood samples were analyzedfor each patient between day -2 and To establish the sensitivity of this assay, we analyzed sinday + 18 after VIP chemotherapy. glecell suspensions of a human small cell lung cancer Preparation and immunostaining on adhesion slides. Mononu(LXFS 428/ 15) and a human breast cancer (MCF-7) xenoclear cells (MNCs) from blood samples were isolated by density cengraft from nude mice in normal peripheral blood or BM trifugation through Ficoll/Hypaque (Pharmacia, Freiburg, GerMNCs. The sensitivity of this assay was 1:4 X lo5, as anamany),washedtwiceinphosphate-buffered saline (PBS), and lyzed by the detection of AE 1/AE3 + KLI and HEA 125 resuspended at 4 to 5 X lo6 cells/mL in PBS. Isolated MNCs were positive human LXFS or MCF-7 tumor cells within normal attached to Poly-L-lysine coated adhesion slides (Fa. Marienfeld, peripheral blood or BM MNCs (Fig1). Bad Mergentheim, Germany) consisting of 3 spots with each 1.4 Before initiation of PBPC mobilization, baseline samples cm in diameter. Routinely, two slides with a total of 6 spots were examined for each patient and for each time point. One hundred from all 46 patients were examined for the presence of cytomicroliters of the cell suspension wasattached on each spot(=4 to keratin and HEA 125-positive circulating cells (Table 1). 5 X IO5 cells/spot). After a 3-minute incubation, the slides were The overall frequency of immunocytochemically positive rinsed with PBS to remove dead cells and cellular debris. Subseblood samples before chemotherapy was 8.7%. In contrast, quently, cells were air-dried for 24 hours and stored at -70’C until BM specimens were found to be cytokeratin- and/or HEAuse. The cells were fixed in aceton/ethanol (10 minutes, 4°C) for positive in 17.4% ofthese patients. subsequent marker analysis. For immunostaining, anticytokeratin Upon VIP chemotherapy G-CSF-induced mobiliza(clone AEI/AE3, IgGI; Boehringer Mannheim, Mannheim, Gertion of PBPCs, cytokeratin- and/or HEA-positive tumor many, and clone KL1, I g G I ; Dianova, Hamburg, Germany) as well cells were recruited in a total of 13 patients (28.3%). In the as anti-epithelial (clone HEA 125, IgGI; Progen, Heidelberg,Germany) monoclonal antibodies (MoAbs) were used. four patientsalready shown to be positive for circulating tuThe anticytokeratin AEI/AE3 antibodies specifically recognize mor cells under steady-state conditions, additional tumor 40- to 56-kD proteins of the acidic as well as all members of the cells were recruited into peripheral blood, with higher total basic subfamily. The KLI antibody recognizes a 56-kD protein of numbers when compared with baseline. In the patients the acidic subfamily. A mixture of both anticytokeratin antibodies without evidence of circulating tumor cells at baseline (n = (AEI/AE3 + KL1) was used. HEA 125 reacts with the 35-kD hu42), tumor cells were recruited in nine patients(2 I .4%). man epithelium-specific cell-surface glycoprotein. The immunoDependent on thepresence or absence of BM infiltration, staining was performed after preincubation with normal serum, acdifferent kinetics of tumor cell mobilization were observed: cording to established procedures (APAAP assay; DAKO, in patients withoutBM disease, tumor cells were mobilized Hamburg, Germany). To increase chromogen intensity, the antionly between days 1 and 7, ie, early after chemotherapy. body reaction was developed twice. Cells containing cytokeratin components or epithelial antigens were stained bright red. Each im- None ofthemhad circulating tumor cells beyond day 9 after 0
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BRUGGER ET AL
638
Relative # of Tumor Cells 100 Expected Value
-1
10 -
m
HEA125
A AE1/3-KL1
-2
10 -3
10 -
B
P
-4
*
10 -
El
-5
10 l
I
I
10-l
1.2
'
I
I
I
-4
IO 3
10
I
105 Ratio Tumor Cells : MNCs
chemotherapy (Table 2). In contrast, in patients with BM infiltration, kinetic analyses displayed a different pattern of tumor cell recruitment, which occurred concomitant to the mobilization of PBPCs, ie, between days 9 and 16 after chemotherapy (Fig 2, Table 2).13Moreover, in three of these patients, tumor cells were also detected early after chemotherapy, ie, between days 1 and 7. Thus, in some patients with BM disease, there was a mobilization of circulating tumor cells in two phases, with a partial clearance between days 5 and 10.
Table 2. Detectionof Tumor Cells UponVIP Mobilization of PBPCs
+ G-CSF-Induced
Patient With Circulatlng Tumor Cells After VIP G-CSF
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Patient
BM-positive (n = 8)
I
Breast 1 Breast 2 Breast 3 Breast 4 Breast 5 SCLC 1 SCLC 2 SCLC 3
BM-negative (n = 38) Breast 6 Breast 7 NSCLC 1 SCLC 4 SCLC 5
Steady State
Between Days 1-7
Between Days 9-1 6
I
1
I
1 o-6
Fig 1. Detectionof epithelial tumor cells within suspensions of peripheral blood MNCs. The sensitivity of the APAAP assay was performed with MCF-7tumorcells that were mixed by serial dilutions with normal peripheral blood MNCs. Data are presented as the relative number of tumor cells as compared with the total cell number per spot.
The overall frequency of tumor cell mobilization in patients without marrow disease was 13% (5 of 38 patients), whereas all patients with BM infiltrating tumors (n = 8) recruited tumor cells after VIP chemotherapy (Table 2). Tumor cells were mobilized only in patients with extensive-disease SCLC ( 5 / I O = 50%),in patients with stage IV breast cancer (7/7 = loo%), as well as in one patient with advanced NSCLC (Table l ) . No cytokeratin- or HEA-positive tumor cells were recruited in patients with limited-disease SCLC, stage II/III breast cancer, or any otheradvanced malignancies after VIP chemotherapy + G-CSF (Table l). The absolute number of recruited tumor cells was highly variable with a broad range of 4 to 5,600 tumor cells per I .6 X IO6 mononuclear cells analyzed. When compared with the absolutewhite blood cell count, these numbers represent a range of less than 3 to more than 8,000 tumor cells per milliliter of whole blood. DISCUSSION
Circulating tumor cells have been detected in patients with neurobla~toma,'~,'~ lymphoma, ovarian cancer, and metastatic breast Our study indicates that the probability of circulating tumor cells under steady-state conditions is much lower when compared with the incidence of BM infiltrating tumor cells, suggesting a lower risk of contamination in peripheral blood, which might argue for the use of PBPCs instead of BM progenitor cells for autotran~plantation.'~ However, because of the low incidence of circulating progenitor cells under steady-state hematopoiesis, progenitor cells have to be mobilized from BM into
STEM
CELLS
MOBILIZATION BLOOD AND OF TUMOR CELLS
A
Fig 2. Pattern of recruitment of malignant epithelialtumor cells in patients without (A) or with (B)BM infiltration. Data are presentedasnumberof HEA and/or cytokeratin-positive tumorcellsper 4 x IO6 mononuclear cells from two patients.
# of Tumor Cells/4xlOe5
B
t of Tumor Cells14xlOe5
0
Days after VIP Chemotherapy
circulation. Thus, the question arises whether the recruitment of PBPCs might also result in a concomitant mobilization of tumor cells. We have shown in thisreport that tumorcell mobilization actually takes place. In all patients with circulating tumor cells under steady-state conditions, the number of tumor cells is increased upon recruitment of PBPCs. In patients with no proof of baseline tumor cells in peripheral blood, the overall detection rate upon mobilization of PBPCs is 21% (9/42 patients), with the highest probability being documented in patients with stage IV breast cancer and extensive-disease small-cell lung cancer. The mechanisms that underlay tumor cell recruitment are unknown. Interestingly, two different patterns of tumor cell mobilization were seen. The first phase of tumor cell recruitment seen early after chemotherapy might represent tumor cells that have been released from rapidly responding tumor areas. The secondpattern of recruitment (corresponding to the time of peak output of progenitor cells) was only observed in patients with BM disease, suggesting that these tumor cells might have been mobilized from the marrow, concomitant with hematopoietic progenitor cells. Possibly, tumor cells that metastasize to BM may share someof the characteristics of hematopoietic progenitor cells, such as homing receptors or adhesion molecules. Recruitment of tumor cells was observed after chemotherapy G-CSF, a procedurethat several groups including our own have shown to be highly effective in mobilizing PBPCS.~.” Therelative contribution of chemotherapy and G-CSF with respect to the mobilization oftumor cells isunclear atthispoint; we are testing inacurrent protocol whether tumor cells are also recruited when G-CSF is administered alone for the mobilization of PBPCs. We have no evidence that tumorcells detected by the immunocytologic method used represent normal epithelial cells which might have been disrupted from normal tissue in conjunction with chemotherapy, because tumor cell mobilization was only observed in breast and SCLC patients.
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639
2
4
6
B
10 12 14 16 18
Days afterVIP Chemotherapy
All other patients who were treated according to the same chemotherapy regimen were negative for tumor cells; however, numbers in the other tumorcategories studied are still too small to permit a definitive conclusion. BM as well as peripheral blood samples from patients with nonmalignant disease are consistently negative both in our own studies as well as inpublished data.20 Clearance of circulating tumor cells was observed after one additional cycle of the same VIP chemotherapy in all SCLC patients, whereas in breast cancer patientstumor cells were still detectable, though in reduced numbers, in two of seven patients. This observation suggests that induction chemotherapy should be administered before PBPCs are mobilized and harvested. However, considering the possibility that tumor cells develop chemoresistance before application of a high-dose regimen, and because quality and quantity of PBPCs decrease with increasing doses of prior chemotherapy and/or radiotherapy,” progenitor cell harvest should notbe delayed too long. The biologic significance of tumor cells contaminating PBPC preparations is unknown. It might be possible that circulating tumor cells primarily reflect advanced stage disease, and long-term prognosis essentially is related to the underlying disease and not to the number of retransfused tumor cells contaminating PBPC harvests. For autologousBM transplantation, it has been shown by Gribben et aI2’in B-cell lymphomas and by Brenner et a12* in childhood acutemyelogenous leukemia (AML)that reinfusion of tumor cells might be an important prognostic indicator in predicting relapse. However, for solid tumors and for autografting with PBPCs, data aremissing. Thus, at present it cannot be estimated whether a positive selection of hematopoietic progenitor cells and/or the depletion of tumor cells from PBPC harvests affects clinical outcome. We would prefer to currently treat patients with PBPCs that have been depleted of contaminating tumor cells, either by retransfusing positively selected CD34’ progenitor ~ e l l s ~ ~ . ~ ~ and/or by ex vivo expanding progenitor cells with the po-
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tential to “biologically purge” tumor cells during ex vivo
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