Tumor Cell Purging by Ex Vivo Expansion of Hemopoietic Stem Cells from Breast Cancer Patients Combined with Targeting ErbB Receptors

Article Outline

• Abstract
• Introduction
• Materials and methods
  • Reagents and Recombinant Human Cytokines
  • Patients and Samples
  • Cell Lines
  • Experimental Contamination Assay
  • Purification of CD34+ Cells
  • Cell Culture Expansion System
  • Hemopoietic Cell Assay
  • Long-Term Culture-Initiating Cells
  • MAM Real-Time Quantitative PCR
  • Statistical Analysis
• Results
  • Ex Vivo Expansion of Purified CD34+ Cells from MPB Derived from Patients with Metastatic Breast Cancer
  • Detection of Tumor Cell Contamination in Unmanipulated and Expanded Leukapheresis Collections Derived from Patients with Metastatic Breast Cancer
  • Effect of Trastuzumab and Gefitinib on Tumor Cells in Coculture
• Discussion
• Acknowledgments
• References
• Copyright

Abstract 

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34⁺ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34⁺ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti–ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34⁺ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both—such as trastuzumab or gefitinib—might increase the efficiency of purging.

Key words:  Ex vivo expansion , Stem cell transplantation , Breast cancer cell purging , Anti-ErbB targeted therapy , Real-time quantitative PCR , Mammaglobin mRNA

Back to Article Outline

Introduction 

The clinical benefit of high-dose chemotherapy programs with autologous stem cell transplantation for the treatment of metastatic breast cancer is a matter of discussion [1, 2]. Studies on the prognostic significance of occult tumor cell contamination in apheresis products show contradictory results and are still not conclusive [3, 4, 5, 6, 7, 8, 9, 10].

Gene-marking studies have shown that in patients with acute or chronic myelogenous leukemia and neuroblastoma, tumor cell reinfusion contributes to relapses after autologous transplantation [11, 12]. Contaminating breast cancer cells in leukapheresis products might be the origin of tumor relapse. Purging of tumor cells from leukaphereses may contribute to increased patient survival. In vitro studies showed that tumor cells in mobilized peripheral blood (MPB) collections are viable and retain clonogenic potential [13, 14, 15, 16]. Ex vivo expansion of progenitor cells might produce a passive elimination of contaminating tumor cells [17]. Recently, we showed that the culture conditions that allow extensive self-maintenance of cord blood–derived hemopoietic progenitors [18] can be extended to MPB in the presence of flt3 ligand (FL), stem cell factor (SCF), thrombopoietin (TPO),interleukin (IL)–6, and serum. Under such culture conditions, transplantable precursor and stem cells still retain their proliferation and multilineage differentiation capacity [19].

ErbB-2 and epidermal growth factor receptor (EGFR) are expressed in human breast carcinoma [20, 21]; they represent ideal targets of anti-ErbB therapies. The humanized anti–ErbB-2 monoclonal antibody trastuzumab has been approved for the treatment of metastatic breast cancer [22, 23, 24, 25]. Similarly, the EGFR tyrosine kinase inhibitor gefitinib (ZR1839) is currently in phase III clinical studies [26, 27, 28, 29].

In this work, we set up a small-scale culture system of MPB from breast cancer patients to obtain the expansion of hemopoietic progenitors and the selective elimination of contaminating tumor cells by targeting ErbB receptors. Because mammaglobin (MAM) is the most specific marker for the detection of epithelial cell contamination in hematologic samples [10, 30], we used MAM messenger RNA (mRNA) amplification by quantitative polymerase chain reaction (PCR) to monitor tumor contamination before and after culture.

Back to Article Outline

Materials and methods 

Reagents and Recombinant Human Cytokines 

Trastuzumab (Herceptin) was obtained from Genentech Inc. (San Francisco, CA). ZD1839 (gefitinib) was kindly provided by AstraZeneca (Macclesfield, UK). Recombinant human (rh) SCF was a generous gift from Amgen (Thousand Oaks, CA), rhIL-3 was obtained from Novartis (Basel, Switzerland), rhIL-6 was obtained from PeproTech (Rocky Hill, NJ), rh-erythropoietin (Eprex) was obtained from Cilag (Milan, Italy), rhFL was kindly provided by S.D. Lyman (Immunex Corp., Seattle, WA), and rhTPO was a generous gift from Kirin Brewery (Tokyo, Japan).

Patients and Samples 

We studied samples from 10 patients with metastatic breast cancer. Informed consent was obtained from all patients according to the institutional guidelines. Patients were evaluated for secondary metastatic lesions by means of chest and abdomen computed tomography and bone scintigraphy.

The characteristics of breast cancer patients are summarized in Table 1. All patients were female. MPB collections from patients with metastatic breast cancer and MPB collections from healthy donors were harvested from the leftovers of leukapheresis procedures. In breast cancer patients, peripheral blood stem cells were collected after recovery from the third course of conventional-dose chemotherapy with paclitaxel (Taxol; Bristol-Myers Squibb, Rome, Italy) 175 mg/m² and vinorelbine (Navelbine; Pierre Fabre, Rome, Italy) 30 mg/m². Human recombinant granulocyte colony-stimulating factor (Granocyte 34; Aventis Pharma, Milan, Italy) 10 μg/kg was administered from day +5 until the completion of stem cell harvest. Healthy donors had received only granulocyte colony-stimulating factor for 5 consecutive days. For the reconstitution experiments of tumor contamination, 12 MPB samples derived from healthy donors were collected after informed consent was given.

Table 1. Characteristics of Enrolled Breast Cancer Patients

Patients were evaluated for secondary metastases by means of chest and abdomen computed tomography and bone scintigraphy.

Cell Lines 

The SKBR3 human breast cancer cell line was routinely maintained in Dulbecco modified Eagle medium (Invitrogen Life Technologies, Gaithersburg, MD) with 10% fetal calf serum (Hyclone, Logan, UT). We selected this cell line on the basis of MAM expression [30], ErbB-2 genomic amplification, and EGFR overexpression [31].

Experimental Contamination Assay 

To set up the contamination model, we performed a reconstitution experiment by using peripheral blood mononuclear cells derived from healthy donors spiked with SKBR3 in limiting dilutions from 1 SKBR3 cell in 10 mononuclear cells to 1 SKBR3 cell in 10⁶ mononuclear cells. We used this experimental model to establish the degree of tumor contamination of leukaphereses from patients.

Purification of CD34⁺ Cells 

CD34⁺ cells were isolated from MPB mononuclear cells with a superparamagnetic microbead selection system by using a high-gradient magnetic field and MiniMACS column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purification efficiency was verified by flow cytometry counterstaining with a CD34 phycoerythrin (Becton Dickinson, San Jose, CA) antibody. In the cell fraction containing purified cells, the percentage of CD34⁺ cells ranged from 90% to 98%.

Cell Culture Expansion System 

Expansion culture was performed in 24-well tissue culture plates with 5 × 10⁵ CD34⁺ cells per well in 1 mL of Iscove modified Dulbecco medium containing 10% calf serum plus SCF (50 ng/mL), FL (50 ng/mL), TPO (10 ng/mL), and IL-6 (10 ng/mL). Total cell number, CD34⁺ cell production, colony-forming cells (CFC), long-term culture-initiating cell (LTC-IC) content, and tumor cell presence were evaluated at the start of culture and after 2 weeks.

To reproduce tumor-contaminated leukapheresis, we performed coculture by spiking 100 SKBR3 cell with 10⁶ CD34⁺ cells derived from healthy donors in the presence of the same growth factors and cultured with or without 30 μg/mL trastuzumab or 2 μmol/L ZR1839 (gefitinib). Fresh antibody and inhibitor were added every 72 hours; growth factors were added every week. After 2 weeks of culture, the cells were analyzed for total cell number, CFC, LTC-IC, CD34⁺, and tumor cell content.

Hemopoietic Cell Assay 

Assays for multilineage granulocyte-macrophage megakaryocyte colony-forming units were performed in plasma clots, as previously described [19]. Briefly, 2 × 10³ CD34⁺ cells were seeded in plasma clots (4 dishes per point) in the presence of IL-3 (20 ng/mL), SCF (50 ng/mL), and erythropoietin (3 U/mL). Colony scoring was performed on day 14 with an optical microscope at ×4 magnitude by counting all the colonies (>50 cells) grown in the plate.

Long-Term Culture-Initiating Cells 

The LTC-IC content of unmanipulated and expanded CD34⁺ cells was analyzed as previously described [19]. Briefly, 2 × 10³ CD34⁺ cells were seeded onto a pre-established irradiated human bone marrow stromal layer and maintained at 37°C for 6 weeks. After the culture period, all cells were harvested and plated for CFC quantitation.

MAM Real-Time Quantitative PCR 

The detection of tumor contamination of MPB collection has been described in detail previously [30]. Briefly, total RNA was used as a template for complementary DNA synthesis, and MAM real-time quantitative PCR was performed with specific primers and TaqMan probes (Perkin Elmer, Foster City, CA) (MAM forward primer, 5′-CAGCGGCTTCCTTGATCCT-3′; MAM reverse primer, 5′-TCATGGTGAGGCTGCTGCT-3′; MAM detection probe, carboxyfluorescein-5′-CCACCCGCGACTGAACACCGA-3′-tetramethylrhodamine). The glyceraldehyde phosphate dehydrogenase (GAPDH) gene was amplified as a control (forward GAPDH primer, 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse GAPDH primer, 5′-GAAGATGGTGATGGGATTTC-3′; GAPDH detection probe, carboxyfluorescein-5′-CAAGCTTCCCGTTCTCAGCC-3′-tetramethylrhodamine). All PCR reactions were performed in triplicate in a 96-well microtiter plate format on an ABI PRISM 7700 Sequence Detector System (Perkin Elmer), and amplification curves were analyzed as previously described [30].

Statistical Analysis 

Each CFC assay was performed in quadruplicate. The results were analyzed by using the Mann-Whitney U nonparametric test. All P values are 2 tailed, with significance set at P < .05. -Fold increases of total cells, CD34⁺ cells, CFCs, and LTC-ICs were calculated, and their means and standard deviations were analyzed.

Back to Article Outline

Results 

Ex Vivo Expansion of Purified CD34⁺ Cells from MPB Derived from Patients with Metastatic Breast Cancer 

We verified the ex vivo expansion potential of our culture system on purified CD34⁺ cells derived from 5 patients with metastatic breast cancer. We analyzed the total cell number, CD34⁺ cell production, and CFC and LTC-IC output after 2 weeks of expansion in the presence of FL, TPO, SCF, and IL-6. Table 2 shows the relative -fold increases of each parameter.

Table 2. Expansion Potential of MPB CD34⁺ Cells Derived from Patients with Metastatic Breast Cancer

A total of 5 × 10⁵ CD34⁺ cells from 5 leukaphereses were cultured as described in “Materials and Methods.” The fold increase of total cells, CD34⁺ cells, CFC, and LTC-IC was evaluated after 2 weeks of expansion.

After 2 weeks of culture, total cells and CD34⁺ cells were expanded 40.8 ± 9.2-fold and 4.2 ± 1.8-fold, respectively. CFCs (12.9 ± 4.0-fold) and LTC-IC (8.7 ± 5.0-fold) were also expanded in all samples. These results were similar to those obtained with CD34⁺ cells derived from healthy donors, as previously demonstrated [19], thus suggesting that chemotherapy does not affect the clonogenic capacity of hemopoietic stem cells in patients with metastatic breast cancer.

Detection of Tumor Cell Contamination in Unmanipulated and Expanded Leukapheresis Collections Derived from Patients with Metastatic Breast Cancer 

We assessed the presence of tumor contamination on mononuclear cells derived from 10 leukaphereses of patients with metastatic breast cancer (Table 1) by MAM quantitative RT-PCR. Five of 10 samples were positive for MAM mRNA. To evaluate the degree of tumor contamination, we compared the MAM amplification curves of positive samples with those obtained on reconstitution experiments with SKBR3 cells and peripheral mononuclear cells from healthy donors. The levels of contamination of leukaphereses from patients were comparable to those obtained in the limiting dilution ranging from 1 SKBR3 cell in 10⁶ to 1 SKBR3 in 10⁵ mononuclear cells of healthy donors (Figure 1).

• 
  • View Large Image
  • Download to PowerPoint

• Figure 1. 

  Quantitation of tumor contamination on MPB harvest. Mammaglobin amplification curves on reconstitution experiments with SKBR3 cells and peripheral mononuclear cells (curves from 1 SKBR3/10 mononuclear cells to 1 SKBR3/10⁶ mononuclear cells from a healthy donor) and on 5 (from patient 1 to patient 5) MPB collections derived from breast cancer patients are shown. NTC indicates no template control. RN indicates reporter fluorescence normalized to internal control.

Leukaphereses from patients were subjected to CD34⁺ cell purification and cultured. After 2 weeks of expansion, the levels of MAM mRNA were compared with those of unmanipulated mononuclear cells and with those of purified CD34⁺. After CD34⁺ selection, our system was unable to detect any level of MAM; this suggests that the tumor load was reduced below the threshold level of detection. During cell culture, 2 different fractions are distinguished: a nonadherent subpopulation, which consists of hemopoietic cells that grow in suspension, and adherent cells containing epithelial tumor cells, monocytes, and endothelial cells. The level of MAM mRNA in these 2 cultured subpopulations was compared with that of unmanipulated mononuclear cells. Tumor marker was increased in the adherent fraction of culture compared with unmanipulated mononuclear cells, thus suggesting that our system expands, also contaminating epithelial tumor cells. Conversely, MAM mRNA was reduced in the suspended fraction in all samples. In 1 sample, tumor cells became undetectable. Table 3 shows the relative amounts of MAM mRNA in each sample (from patient 1 to patient 5) after each step of in vitro manipulation compared with those of a standard calibration curve with SKBR3 and mononuclear cells from healthy donors.

Table 3. Relative Amounts of MAM mRNA in MPB from Breast Cancer Patients (from Patient 1 to Patient 5) Compared with Those of the Calibration Curve

Sample⁎	Calibration Curve†	Before CD34 Selection	After CD34 Selection	Adherent Fraction After Expansion	Nonadherent Fraction After Expansion
1		1.25	ND	2.51	0.53
2		9.87	ND	58.26	1.55
3		2.67	ND	4.16	ND
4		0.71	ND	8.56	0.4
5		1.86	ND	4.47	0.09
Calibration
1:106	1
10:106	4.8
100:106	90

ND indicates not detectable.

Effect of Trastuzumab and Gefitinib on Tumor Cells in Coculture 

It has been demonstrated that trastuzumab, a monoclonal antibody directed against ErbB-2, inhibits the proliferation of ErbB-2–overexpressing cells [22, 23, 24, 25, 32]. Similarly, ZD1839, a tyrosine kinase inhibitor specific for EGFR, is able to block the growth of EGFR-expressing human cancer cells [26, 27, 28, 29]. We analyzed whether these 2 molecules might contribute to purge contaminated stem cell collections during in vitro manipulation.

We performed coculture with CD34⁺ cells obtained by leukaphereses from healthy donors and the SKBR3 cell line in the presence or absence of trastuzumab or gefitinib. This cell line was selected for the characteristics of ErbB-2 genomic amplification and EGFR overexpression, which allow trastuzumab and gefitinib to inhibit cell growth. After 2 weeks of culture in the presence of trastuzumab, MAM mRNA was undetectable in the suspended fraction (data not shown). Relative quantitation with comparative threshold cycle methods of the tumor marker (Table 4) demonstrated that on adherent fractions, the levels of MAM in the trastuzumab-treated cells (5.25-fold increase of MAM with respect to the start of culture) were lower than those in untreated cultures (278.2-fold increase of MAM with respect to the start of culture), which presented transcript levels 52.9-fold higher than in the presence of the antibody. Figure 2 shows MAM and GAPDH amplification curves obtained after 2 weeks of coculture in reconstitution assays with SKBR3 cells and hemopoietic precursors in the absence or presence of trastuzumab. The same results were obtained upon EGFR tyrosine kinase inhibitor treatment. Moreover, by investigating CFC content, we demonstrated that trastuzumab and gefitinib did not change the clonogenic capacity of hemopoietic cells (Table 5).

Table 4. Relative Quantitation of Mammaglobin by Using the Comparative Ct Methods

Variable	MAM Average Ct	GAPDH Average Ct	ΔCt (MAM−GAPDH)	ΔΔCt (ΔCt− DCtSKBR3 T0)	MAM (Relative to SKBR3 T0)
SKBR3 T0	33.65±0.002	28.66±0.12	4.99	0	1
SKBR3 + T	31.23±0.11	28.63±0.44	2.6	−2.39	5.25
SKBR3 − T	25.34±0.46	28.47±0.34	−3.13	−8.12	278.2

Ct indicates the threshold cycle, defined as the PCR cycle at which the reporter fluorescence dye cleaved from the MAM probe generates a target-specific detection signal (ΔRn) that passes above the baseline. The ΔCt value is determined by subtracting the average GAPDH Ct value from the average MAM Ct value. The calculation of ΔΔCt involves subtraction by the ΔCt calibrator value (SKBR3 T₀). The range given for MAM relative to SKBR3 T₀ is determined by evaluating the expression 2^−DDCt

SKBR3 T₀ indicates the start of coculture of CD34⁺ cells obtained from leukaphereses of healthy donors spiked with SKBR3 cells.

SKBR3 + T indicates coculture of CD34⁺ cells obtained from leukaphereses of healthy donors spiked with SKBR3 cells and grown for 2 weeks in the presence of trastuzumab.

SKBR3 − T indicates coculture of CD34⁺ cells obtained from leukaphereses of healthy donors spiked with SKBR3 cells and grown for 2 weeks in the absence of trastuzumab.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 2. 

  Effect of trastuzumab on cocultures of SKBR3 cells and hemopoietic precursor cells. Representative amplification curves are shown of GAPDH (control gene) and MAM (target gene) obtained by mammaglobin (MAM) quantitative reverse transcriptase-PCR in coculture of SKBR3 tumor cells and normal progenitor cells. Cells were analyzed before (T0) and after 2 weeks in the presence (+T) or absence (−T) of trastuzumab. NTC indicates no template control. RN indicates reporter fluorescence normalized to internal control.

Table 5. Colony-Forming Cell (CFC) Content

Variable	CFC
−Trastuzumab	182(64–346)
+Trastuzumab	147(52–233)
−Gefitinib	122(70–154)
+Gefitinib	136(78–148)

Tumor-contaminated leukaphereses were reconstituted by spiking 100 SKBR3 cells with 10⁶ CD34⁺ cells derived from healthy donors and expanded with or without 30 μg/mL trastuzumab and 2 μmol/L ZR1839. After the period of culture, CFC content was evaluated. Values indicate the median and range of several experiments (6 experiments with different donors in the presence of trastuzumab and 5 with gefitinib).

Back to Article Outline

Discussion 

This work proposes a novel strategy for tumor cell purging of leukaphereses from patients with metastatic breast cancer scheduled for high-dose chemotherapy programs with hemopoietic stem cell rescue. This system is based on ex vivo expansion of CD34⁺ cells from patients with metastatic breast cancer in combination with ErbB receptor targeting.

In our laboratory, we previously set up an efficient expansion method of adult CD34⁺ cells derived from healthy donors [19]. Here we report that this culture condition (FL, TPO, SCF, and IL-6 in the presence of serum) allows the maintenance and even some expansion of CD34⁺ derived from tumor cell–positive collections. The absolute number of CD34⁺ cells and CFC and LTC-IC content were significantly increased after 2 weeks of culture. The hemopoietic expansion potential in contaminated collections was not different from that of healthy donors [19]. Nonetheless, tumor cells persist and proliferate in vitro, as demonstrated by quantitative reverse transcriptase-PCR at the end of the culture period. However, we observed differences between the adherent and suspended fractions of culture: the adherent cells contained higher levels of MAM mRNA than unmanipulated cells, whereas on the suspended fraction, which contains most hematopoietic progenitors, there was a reduction of the marker level in all cultured samples. The ability of tumor epithelial cells to adhere on plastic could contribute to decreasing the tumor contamination. Positive selection of CD34⁺ cells from MPB of breast cancer patients is not enough to completely eliminate epithelial tumor cells in hemopoietic autografts, and the ex vivo culture condition does not result in an effective purging of tumor cells. For these reasons, to improve the efficiency of the tumor-purging protocol, we considered additional strategies, such as targeting ErbB family receptors during hemopoietic ex vivo expansion. We investigated whether trastuzumab, the monoclonal antibody against ErbB-2 [22, 23, 24, 25, 31], and the EGFR tyrosine kinase receptor inhibitor ZD1839 (gefitinib) [26, 27, 28, 29] are able to reduce the tumor load without affecting the clonogenic potential of hemopoietic cells.

CD34⁺ cells obtained from leukaphereses of healthy donors were mixed with the ErbB-2⁺ and EGFR⁺ SKBR3 human breast cancer cell line and cultured for 2 weeks in the presence of trastuzumab and gefitinib. After 2 weeks of culture in the presence of trastuzumab, the tumor marker was undetectable in suspended cells, and there was a 52.9-fold reduction in the adherent fraction. Moreover, clonogenic assays showed that trastuzumab and gefitinib spare hemopoietic cells. These results demonstrate that EGFR targeting does not upset the hemopoietic developmental capacity.

In conclusion, our protocol might be used as an efficient in vitro purging of leukapheresis collections from breast cancer patients with ErbB-2 overexpression or EGFR expression.

Back to Article Outline

Acknowledgments 

This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro 2004-2006 (Milan, Italy), from Progetto Consiglio Nazionale delle Ricerche; Ministero dell’Istruzione, dell’Università e della Ricerca; Progetto Strategico Oncologia 5, and from the Ministero dell’Istruzione, dell’Università e della Ricerca (Progetto Ministero dell’Istruzione, dell’Università e della Ricerca; ex 60% 2000; F.L., G.C., M.G., Y.P., W.P., and M.A.).

Back to Article Outline

References 

1. Nieto Y . The verdict is not in yet. Analysis of the randomized trials of high-dose chemotherapy for breast cancer . Haematologica . 2003;88:201–211
   • View In Article
   • MEDLINE
2. Nieto Y , Jones RB , Shpall EJ . High-dose chemotherapy for breast cancer (is another look warranted?) . Curr Opin Oncol . 2004;16:114–119
   • View In Article
   • MEDLINE
   • CrossRef
3. Solano C , Badia B , Lluch A , et al.   Prognostic significance of the immunocytochemical detection of contaminating tumor cells (CTC) in apheresis products of patients with high-risk breast cancer treated with high-dose chemotherapy and stem cell transplantation . Bone Marrow Transplant . 2001;27:287–293
   • View In Article
   • MEDLINE
   • CrossRef
4. Syme R , Stewart D , Rodriguez-Galvez M , et al.   Micrometastases in apheresis products predicts shorter progression-free and overall survival in patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous blood stem cell transplantation (ABSCT) . Bone Marrow Transplant . 2003;32:307–311
   • View In Article
   • MEDLINE
   • CrossRef
5. Patriarca F , Sacco C , Sperotto A , et al.   Prognostic significance of the detection of tumour cells in peripheral blood stem cell collections in stage II and III breast cancer patients treated with high-dose therapy . Bone Marrow Transplant . 2003;31:789–794
   • View In Article
   • MEDLINE
   • CrossRef
6. Reed E , Kessinger A , Murphy B , Tarantolo S , Traystman M , Sharp JG . Occult tumor cells detected in autologous blood stem cell harvests have no impact on 5 year outcomes for breast cancer patients with 4-9 positive nodes treated with adjuvant high-dose therapy and stem cell transplantation . Bone Marrow Transplant . 2003;31:571–574
   • View In Article
   • MEDLINE
   • CrossRef
7. Viret F , Chabannon C , Sainty D , et al.   Occult tumor cell contamination in patients with stage II/III breast cancer receiving sequential high-dose chemotherapy . Bone Marrow Transplant . 2003;32:1059–1064
   • View In Article
   • MEDLINE
   • CrossRef
8. Pecora AL , Lazarus HM , Jennis AA , et al.   Breast cancer cell contamination of blood stem cell products in patients with metastatic breast cancer (predictors and clinical relevance) . Biol Blood Marrow Transplant . 2002;8:536–543
   • View In Article
   • Abstract
   • Full-Text PDF (129 KB)
   • CrossRef
9. Nieto Y , Franklin WA , Jones RB , et al.   Prognostic significance of occult tumor cells in the apheresis products of patients with advanced breast cancer receiving high-dose chemotherapy and autologous hematopoietic progenitor cell support . Biol Blood Marrow Transplant . 2004;10:415–425
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (161 KB)
   • CrossRef
10. Ferrucci PF , Rabascio C , Mazzetta C , et al.   Mammaglobin expression in leukapheresis products is a predictive marker of poor prognosis in women with high-risk breast cancer . Clin Cancer Res . 2004;10:6039–6046
    • View In Article
    • MEDLINE
    • CrossRef
11. Brenner MK , Rill DR , Moen RC , et al.   Gene marking to trace origin relapse after autologous bone marrow transplantation . Lancet . 1993;341:85–86
    • View In Article
    • Abstract
    • CrossRef
12. Deisseroth AB , Zu Z , Claxton D , et al.   Genetic marking shows that Ph⁺ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow transplantation for CML . Blood . 1994;3:3068–3076
    • View In Article
13. Kasimir-Bauer S , Mayer S , Bojko P , Borquez D , Neumann R , Seeber S . Survival of tumor cells in stem cell preparations and bone marrow of patients with high-risk or metastatic breast cancer after receiving dose-intensive or high-dose chemotherapy . Clin Cancer Res . 2001;7:1582–1589
    • View In Article
    • MEDLINE
14. Rill DR , Santana VM , Roberts WM , et al.   Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells . Blood . 1994;84:380–383
    • View In Article
    • MEDLINE
15. Ross AA , Cooper BW , Lazarus HM , et al.   Detection and viability of tumor cells in peripheral blood stem cell collection from breast cancer patients using immunocytochemical and clonogenic assay techniques . Blood . 1993;82:2605–2610
    • View In Article
    • MEDLINE
16. Passos-Coelho JL , Ross AA , Kahn DJ , et al.   Similar breast cancer cell contamination of single-day peripheral-blood progenitor-cell collections obtained after priming with hematopoietic growth factor alone or after cyclophosphamide followed by growth factor . J Clin Oncol . 1996;9:2569–2575
    • View In Article
17. Lundell BI , Vredenburgh JJ , Tyer C , DeSombre K , Smith AK . Ex vivo expansion of bone marrow from breast cancer patients (reduction in tumor cell content through passive purging) . Bone Marrow Transplant . 1998;22:153–159
    • View In Article
    • MEDLINE
18. Piacibello W , Sanavio F , Garetto L , et al.   Extensive amplification and self-renewal of human primitive hemopoietic stem cells from cord blood . Blood . 1997;89:2644–2653
    • View In Article
    • MEDLINE
19. Gammaitoni L , Bruno S , Sanavio F , et al.   Ex-vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution . Exp Hematol . 2003;31:261–270
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (419 KB)
    • CrossRef
20. Salomon DS , Brandt R , Ciardiello F , Normanno N . Epidermal growth factor-related peptides and their receptors in human malignancies . Crit Rev Oncol Hematol . 1995;19:183–232
    • View In Article
    • Full-Text PDF (6802 KB)
    • CrossRef
21. Prenzel N , Fischer OM , Streit S , Hart S , Ullrich A . The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification . Endocr Relat Cancer . 2001;8:11–31
    • View In Article
    • MEDLINE
    • CrossRef
22. Carter P , Presta L , Goran CM , et al.   Humanization of an anti-p185HER2 antibody for human cancer therapy . Proc Natl Acad Sci U S A . 1992;89:4285–4289
    • View In Article
    • MEDLINE
    • CrossRef
23. Pegram MD , Finn RS , Arzoo K , Beryt M , Pietras RJ , Slamon DJ . The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells . Oncogene . 1997;15:537–547
    • View In Article
    • MEDLINE
24. Tokuda Y , Ohnishi Y , Shimamura K , et al.   In vitro and in vivo anti-tumour effects of a humanized monoclonal antibody against c-erbB-2 product . Br J Cancer . 1996;73:1362–1365
    • View In Article
    • MEDLINE
25. Baselga J , Norton L , Albanell J , Kim YM , Mendelsohn J . Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts . Cancer Res . 1998;58:2825–2831
    • View In Article
    • MEDLINE
26. Baselga J . New technologies in epidermal growth factor receptor-targeted cancer therapy . Signal . 2000;1:12–21
    • View In Article
27. Ciardiello F , Caputo R , Bianco R , et al.   Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Gefitinib), an epidermal growth factor receptor-selective tyrosine kinase inhibitor . Clin Cancer Res . 2000;6:2053–2063
    • View In Article
    • MEDLINE
28. Albanell J , Cordony-Servat J , Rojo F , et al.   Activated extracellular signal-regulated kinase (association with epidermal growth factor receptor/transforming growth factor α expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments) . Cancer Res . 2001;61:6500–6510
    • View In Article
    • MEDLINE
29. Albanell J , Rojo F , Averbuch S , et al.   Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients (histopathologic and molecular consequences of receptor inhibition) . J Clin Oncol . 2002;20:110–124
    • View In Article
    • MEDLINE
    • CrossRef
30. Leone F , Perissinotto E , Viale A , et al.   Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction . Bone Marrow Transplant . 2001;27:517–523
    • View In Article
    • MEDLINE
    • CrossRef
31. Wels W , Beerli R , Hellmann P , et al.   EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins . Int J Cancer . 1995;60:137–144
    • View In Article
    • MEDLINE
    • CrossRef
32. Sliwkowski MX , Lofgren JA , Lewis GD , Hotaling TE , Fendly BM , Fox JA . Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin) . Semin Oncol . 1999;26:60–70
    • View In Article
    • MEDLINE