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Response of Circulating Tumor Cells to Systemic Therapy in Patients With Metastatic Breast Cancer: Comparison of Quantitative Polymerase Chain Reaction and Immunocytochemical Techniques

From the Cancer Research Campaign Laboratories, Division of Cancer Cell Biology, and Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital; and Department of Surgery, Charing Cross Hospital, London, United Kingdom.

Address reprint requests to Martin J. Slade, PhD, Cancer Research Campaign Laboratories, Division of Cancer Cell Biology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd, London W12 ONN, United Kingdom; email m.slade{at}ic.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: We previously developed a quantitative system for the detection of cytokeratin 19 (CK-19) transcripts using reverse transcriptase polymerase chain reaction (PCR) to detect breast carcinoma cells in blood and bone marrow. The aim of this study was to determine the value of this system in monitoring patients with metastatic disease and to compare it with an established immunocytochemical method.

PATIENTS AND METHODS: Patients with progressive, locally advanced, and metastatic breast cancer (all stage IV) who were due to start systemic treatment were recruited. Blood samples were analyzed for CK-19 transcripts using quantitative PCR (QPCR) and immunocytochemistry (ICC) throughout their course of treatment.

RESULTS: One hundred forty-five blood samples were obtained from 22 patients over 13 months. Seventy-two (49.6%) of these samples were positive by QPCR, and 56 (42%) of 133 were positive by ICC. Of the 133 specimens analyzed by both techniques, 95 (71.4%) had the same results for each, and of the 71 samples that were positive, 40 (56%) were positive by both methods. The relationship between the number of cells detected and the QPCR values was statistically significant (P < .0001). Of the 25 courses of assessable treatment, 17 (68%) of 25 treatment outcomes (either response or disease progression) were reflected by QPCR measurements, and 12 (57%) of 21 were reflected by ICC. During the course of the study, five patients showed a response, and of these, ICC was in agreement in four cases (80%) and QPCR in three cases (60%). Eighteen courses of treatment resulted in progression of the disease; however, only 15 of these were assessable by ICC. ICC was in agreement in eight (53%) of 15 of these cases, and QPCR in 15 (83%) of 18 cases.

CONCLUSION: Circulating carcinoma cells are frequently found in patients with metastatic breast cancer. In the majority of patients, cancer cell numbers as evaluated by QPCR or ICC reflected the outcome of systemic treatment.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PREVIOUS STUDIES have shown that the presence of micrometastases in the bone marrow of early (stage I and II) breast cancer patients, as evidenced by immunocytochemical markers, is correlated with other prognostic indicators for early relapse, such as tumor size, grade, lymph-vascular invasion, and local lymph node infiltration,^1-3 and with overall survival.^2,4 Unfortunately, little is known about the natural history of micrometastases, and it has been suggested by many groups that the monitoring of this minimal residual disease (MRD) could be used to improve disease staging, as a marker for evaluating new therapeutic strategies, and to assess treatment response in individual patients.

Most current methods do not seem to be sensitive enough to detect circulating cells in significant numbers of patients with early-stage carcinomas.^5-7 However, this may be due not only to inadequate sensitivity of the assays used but also to the fact that such cells may only appear periodically in the circulation during early tumor development. There may be intermittent shedding of tumor cells into the circulation corresponding with microinvasive events within the tumor. Previous studies have shown detection rates of MRD in the peripheral blood of patients with solid tumors in the order of 0% to 27% using polymerase chain reaction (PCR) methodology^7,8 and 0% to 5% using immunocytochemistry (ICC)^8,9 in patients with early-stage disease. Patients with advanced disease have a much greater tumor burden and are, therefore, more likely to have tumor cells present at blood sampling and as such represent a group of patients who can be studied to determine the effect of therapy on the number of circulating tumor cells. Nested reverse transcriptase PCR (RT-PCR) seems to have a greater sensitivity than ICC in the detection of isolated tumor cells both in vitro and in patient samples,^7,8 but the specificity of RT-PCR for cytokeratin 19 has been brought into question.^10,11 To try to overcome this problem, we developed a new quantitative nested RT-PCR for CK-19, with the number of transcripts expressed as a ratio as compared with the Abelson oncogene (ABL).¹² This allows for a distinction between background levels of CK-19 mRNA and higher levels that are found in patients with breast cancer. ABL is used as the reference gene, because unlike, for example, actin and glyceraldehyde-3-phosphate dehydrogenase, there are no ABL pseudogenes.

The aim of this study was two-fold. First, we wished to evaluate the impact of conventional chemotherapy on the presence of circulating peripheral-blood tumor cells in patients with advanced breast cancer and to determine whether the presence, absence, or changes in numbers of these cells using quantitative PCR (QPCR) corresponded with clinical course. Second, we wished to further evaluate our new QPCR methodology and compare it with conventional ICC.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
All eligible patients commencing systemic therapy over a set period were included in the study. Because the main aim was to determine the relationship between response (using International Union Against Cancer [UICC] criteria of tumor response ¹³ ) and circulating tumor cells, we primarily included patients commencing treatment with cyclophosphamide, methotrexate, and fluorouracil (CMF) or with fluorouracil, epirubicin, and cyclophosphamide (FEC) chemotherapy or docetaxel and aromatase inhibitors. All patients had stage IV disease and attended Charing Cross Hospital Oncology Department for treatment of advanced breast cancer between June 1997 and August 1998. Inclusion criteria were (1) histologically or cytologically proven progressive metastatic disease, (2) clinically or radiologically assessable disease, and (3) no treatment with chemotherapy for advanced disease in the past 4 months. No patients had received any previous endocrine or other treatment for at least 1 week before the blood samples were taken. All patients were clinically examined and had chest radiology, liver computed tomography, and bone scan with radiologic confirmation of any areas of increased tracer uptake before entering the study.

The study was reviewed and approved by the Riverside Ethics Review Board, and all patients gave informed consent to the study before blood samples were taken. Samples were obtained before the start of chemotherapy and then at 3- to 4-week intervals, within 48 hours of the beginning of the next cycle of chemotherapy. Assessment of treatment response, according to UICC criteria,¹³ was made by clinicians who were blind to the assay results. Those undertaking the QPCR and ICC were blind to the clinical status and identity of the patients.

Treatment Dosage and Schedules
Patients received chemotherapy as follows: for CMF, cyclophosphamide 600 mg/m², methotrexate 40 mg/m², and fluorouracil 600 mg/m² were administered on days 1 and 8 of a 4-week cycle; epirubicin was given as 50 mg/m² on days 1 and 8 of a 4-week cycle. For FEC, fluorouracil 600 mg/m², epirubicin 50 mg/m², and cyclophosphamide 600 mg/m² were administered on days 1 and 5 (fluorouracil and cyclophosphamide on day 8) of a 4-week cycle; docetaxel 100 mg/m² was administered every 3 weeks. Regarding endocrine therapy, patients received tamoxifen 20 mg/d, formestane 250 mg every 2 weeks, and anastrozole 1 mg/d. Treatments were continued for up to 6 months according to clinical response and toxicity. Patients with complete or partial response continued full courses of treatment. Those with stable disease continued with treatment until disease progression occurred. Patients with progressive disease who were receiving therapy were changed to a different regimen.

Treatment Response
Response was defined in accordance with standard UICC criteria¹³ as follows: complete response was defined as the complete disappearance of all clinical disease by physical or radiologic examination for a period of at least 4 weeks. Partial response was defined as a greater than 50% reduction of tumor size as determined by two measurements not less than 4 weeks apart. Stable disease was defined as less than 25% progression or less than 50% tumor reduction, and progressive disease was defined as a greater than 25% increase in tumor measurements at any time.

Preparation of Blood and Samples
A 20-mL blood sample was collected in 10-mL Vacutainers (Becton Dickinson, Cowley, United Kingdom) to which 150 U of preservative-free heparin had been added (Leo Laboratories, Risborough, United Kingdom). To minimize the risk of skin contamination, at least 10 mL of blood was taken for routine complete blood cell count and biochemical screening.

Blood samples were prepared as previously described.¹² Briefly, the mononucleocytes were separated from the blood over Ficoll-Paque (Pharmacia, St. Albans, United Kingdom). The interface cells were then removed and washed, and the RBCs were removed using a lysis buffer followed by a repeated wash. The mononuclear cells were then counted and aliquoted for QPCR and ICC on the basis of at least 2.5 x 10⁶ cells for each methodology. The cell pellet was resuspended in guanidine thiocyanate for PCR and in phosphate-buffered saline for ICC.

ICC
Cells were cytocentrifuged onto glass slides at 110 x g for 5 minutes using a 30-F cytocentrifuge (Hettich, Tuttlingen, Germany) at a concentration of 5 x 10⁵ per area (240 mm²). Staining was carried out in accordance with previously described procedures.¹⁴ We have previously used this methodology successfully, detecting no false-positives in 45 control blood samples and 15 control bone marrow specimens. The primary antibody (A45-B/B3; Micromet, Munich, Germany) was used at a concentration of 2 mg/mL, followed by the bridging secondary antibody, (Z259; Dako, Hamburg, Germany). Finally, the alkaline phosphatase antialkaline phosphatase (APAAP) complex was added (D651; Dako), and the reaction was developed with new fuscin. An isotype immunoglobulin G1 mouse myeloma antibody, MOPC21 (Sigma, Deisenhofen, Germany), served as the negative control. The cytospins where then screened for positive events without using a counterstain. The malignant breast cell line MCF-7 was used as a positive control. The MCF-7 cells were maintained in DMEM with 10% fetal calf serum and penicillin (100 U/mL), streptomycin (0.1 mg/mL), and glutamine (2 mmol/L).

RT-PCR
Synthesis of cDNA was performed as described previously.¹⁵ Samples were tested initially for CK-19 by nested PCR followed by quantitation on all positive samples as previously described.¹² Briefly, 5 µL of cDNA (equivalent to a median of approximately 10⁶ cells) was mixed with 20 µL of first-step mix; PCR was then performed at 30 cycles of 96°C for 1 minute, 69°C for 25 seconds, and 72°C for 1 minute, followed by a 10-minute extension at 72°C. Product from this reaction was reamplified with internal primers; 19 µL of second-step mix was mixed with 1 µL of first-step reaction product, and amplification was performed using the same cycling conditions as above. Rigorous precautions were taken to prevent contamination by PCR product carryover. All pre-PCR manipulations were performed in a PCR-only designated laminar flow cabinet and using plugged pipette tips. At least two negative controls were included per run; these were prepared last. The reaction products were electrophoresed on a 1.8% agarose gel in a separate room using dedicated pipettes. A band of 463 base pairs (bp) was visualized for CK-19–positive samples. Ethidium bromide–stained gels were deemed to be sufficiently sensitive, as Southern blot analysis would only have served to increase the sensitivity of both QPCR methodologies, thus resulting in the same ratio.

QPCR
Competitive PCR was performed as previously described.^12,16 Briefly, nested PCR for CK-19 was performed as above, except that a titration series of independent reactions containing 2.5 µL of cDNA plus 2.5 µL of known competitor dilution were added to 20 µL of first-step mix instead of 5 µL of cDNA. To improve the clarity of the bands, first-step PCRs were diluted 400-fold in water and 1 µL was used to seed the second-step reaction. The competitor PCR product was seen at 588 bp, and equivalence points were estimated by inspection.

Quantification of ABL transcripts as an internal control for the amount and quality of cDNA¹⁷ was performed for all samples by a single-step PCR: 2.5 µL of cDNA plus 2.5 µL of competitor dilution were added to 20 µL of ABL mix. Bands were visualized at 385 bp for the ABL gene and at 486 bp for the competitor.

QPCR results were expressed as the ratio of CK-19:ABL for specimens that were positive for CK-19. Samples that were negative for CK-19 were expressed as negative:(the number of ABL transcripts detected in the same volume of cDNA).

From a series of 45 control blood samples, we had previously established a CK-19:ABL ratio of 1 CK-19:1,000 ABL transcripts to be the figure above which samples were regarded as negative; conversely, samples with a ratio of less than 1:1000 (ie, 1:999 to 1:1) were deemed positive.¹² The equivalence point was determined visually, which we have previously shown has a low variation (17%) with an experienced operative.¹⁶

Statistical Methods
A Spearman rank test was used to describe the quantitative relationship between the numbers of cells detected by ICC and the ABL values across all samples. For CK:ABL ratios greater than 1:1000 (ie, negative), the ABL values were included in the statistical calculation, although the values are not shown in the results table. To measure the correlation between the qualitative results for ICC and PCR and the relationship between PCR and ICC with the clinical outcome, agreement was measured using kappa values, with the agreement strength being described as modified from Landis and Koch.¹⁸ For statistical analysis of the four groups as described by numbers of cells detected by ICC (0, < five, five to nine, > nine cells), a one-way analysis of variance was used to see if there were global differences in the four groups. However, the required assumption of normal distribution residuals was not valid, and the nonparametric Kruskal-Wallis test was used instead. Contrasts were also performed, each group was compared with each other group, and the group with 0 cells on ICC was compared with the other groups as a whole. The contrasts were obtained using the Mann-Whitney U test and corrected for multiple comparisons by Bonferroni adjustment.

	RESULTS

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Concordance
Of the 22 patients in this study, 17 (77.3%; patient nos. 1 through 8, 10, 11, 13, 15, and 17 through 21) showed concordance between the two methodologies. From a total of 25 assessable chemotherapeutic treatment cycles, 17 (68%) showed clinical changes that were mirrored by changes in disease load as measured by QPCR (kappa = 0.43; moderate agreement), and 12 (57%) of 21 showed clinical changes using ICC (kappa = 0.38; fair agreement).

General
A total of 145 blood samples were obtained from 22 patients over a period of up to 13 months. Details are listed in Table 1 (patient nos. 8 through 22). Patient clinical characteristics are listed in Table 2. Four of 22 patients had local disease only with no evidence of distant metastases on scanning or radiology. All specimens were available for QPCR, with 139 samples also available for ICC (six blood samples had inadequate mononucleocyte numbers for ICC). Six specimens were positive using the isotype control antibody on ICC, and so these results were impossible to interpret.

Response
Six courses of systemic treatment were given to the four patients with locally advanced disease (patient nos. 1 through 4; data not shown) during the time when micrometastases were being monitored on 23 occasions. Three of the four patients showed a single positive cell by ICC, and one showed a weakly positive result (1:800) by QPCR. In view of the fact that this occurred on only one occasion in each patient, we concluded that this finding (ie, a transient positive cell or weakly positive QPCR result) is unlikely to be of significance in this setting. The remaining 19 samples were negative by both methodologies (82% concordance between results).

Three (16.6%) of the 18 patients (patient nos. 5 through 7) with systemic overt metastases, who had four treatment cycles, had negative results in the same pattern as those with locally advanced disease (two patients had a single positive cell on one occasion on ICC; data not shown). Consequently, we were unable to assess treatment response with micrometastatic status in those patients.

A total of 104 blood samples were taken from the remaining 15 patients (patient nos. 8 through 22), and 72 (69%) were positive using QPCR. ICC on the same blood samples was positive in 50 (53%) of 94 specimens. From the same patient group, 61 (64.8%) of 94 specimens showed the same results on QPCR as compared with ICC. Of the 33 samples with differing results between the two techniques, 23 (69.6%) were positive using QPCR but were negative on ICC analysis, indicating the greater sensitivity of QPCR.

In the remaining 15 patients (patient nos. 8 through 22), 72 (69%) and 50 (53%) of the samples were positive using QPCR and ICC, respectively. Seventeen (68%) of 25 assessable chemotherapeutic treatment cycles showed clinical changes that were mirrored by changes in disease load as measured by QPCR, and 12 (57%) of 21 using showed clinical changes as measured by ICC (Table 1).

During the study, five patients showed evidence of response; four of five were assessable by ICC and three of five were assessable by QPCR (Figs 1 A and 1D). Progressive disease was seen in 12 patients, and of these, nine showed an increase in disease load by QPCR (Figs 1E and 1F; patient nos. 9 through 14 and 17 through 19), and five of nine patients showed an increase in disease load by ICC (Figs 1B and 1C; patient nos. 10, 11, and 17 through 19). Eighteen courses of treatment resulted in progression of the disease (only 15 of which were assessable by ICC); ICC was in agreement in eight (53%) of these cases, and QPCR was in agreement in 15 (83%) (Table 3).

View larger version (33K): [in this window] [in a new window]   Fig 1. (A) ICC results of patients showing response; patient no. 14 (3) had a negative result. (B) and (C) ICC results of patients showing clinical progression; patient nos. 9, 14 (2), 20 (1), and 22 (2) had negative results; nos. 13 (1) and 13 (2) had isotype-positive results, and nos. 12 and 22 (1) had no sample. (D) QPCR results of patients showing response; patient nos. 8 and 14 (3) had negative results. (E) and (F) QPCR results of patients showing clinical progression. Numbers in parentheses indicate the treatment course (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

When assessing numbers of cells, there was a significant inverse correlation (P < .0001) with QPCR values when considering all samples. However, when examining samples from an individual, there did not always seem to be a relationship between the CK-19:ABL ratio and cell numbers stained on ICC. There may be several reasons for this. First, the antibody used was a pancytokeratin antibody that detects CK-8, CK-18, and CK-19, and second, because of tumor heterogeneity and the small numbers of metastatic cells detected, levels of expression of CK-19 may vary widely between cells in the same individual. Finally, due to the broad-spectrum coverage of the antibody, comparisons of staining intensity and expression levels of CK-19 mRNA could not be made; in all positive samples, all cells were intensely stained.

Changes in circulating tumor cell levels in patients with systemic disease who had detectable disease using QPCR and ICC reflected the clinical progression/regression of the disease in a majority of patients, which is in agreement with other groups that have studied smaller numbers of patients.^20,21 Although the absolute values did not always reflect the clinical course at a single point during therapy, the trend over a period of months was more important. QPCR also reflected the clinical outcome more frequently than did ICC. The clinical importance of circulating CK-19–positive cells is at present still not definitely proven; however, we have investigated the presence of plasma DNA and loss of heterozygosity and microsatellite instability in many of these patients and related this back to the original tumor (Shaw et al, manuscript submitted for publication). The CK-19–positive detectable cells are identical in their cytologic characteristics to the cells in bone marrow. In this context, the presence of the cells carries a poor prognosis, even 12 years after diagnosis.²²

We conclude that it is possible to monitor disease response from peripheral-blood samples using QPCR in patients with metastatic breast cancer, because our results indicate that circulating tumor cell levels reflect changes in disease load. Previous studies have confirmed that the presence of MRD in the bone marrow of primary breast cancer patients indicates an increased risk of relapse.^2,3,23 We have shown that it is possible to equate disease response with circulating tumor burden using a QPCR assay. Therefore, although these results are somewhat preliminary, it may be possible to apply such an assay to monitor patients who are considered to be disease-free. As mentioned previously, we are currently undertaking a project to monitor a large number of patients who are being followed-up after primary surgery and adjuvant therapy using bone marrow aspirates. We have currently analyzed and compared QPCR and ICC in 100 patients with primary breast cancer and on follow-up for a total of 450 samples. Concordance rates in this study are comparable to the ones presented in this article. Such a monitoring system could be used as a surrogate marker in identifying patients who are at increased risk of relapse over a prolonged follow-up period. Patients who are found to have an increasing CK-19:ABL ratio could be candidates for further systemic adjuvant therapy.

	ACKNOWLEDGMENTS

 
Supported by the Cancer Research Campaign, North Thames Regional Health Authority, and the Leukaemia Research Fund.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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12. Slade MJ, Smith BM, Sinnett HD, et al: Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 17:870-879, 1999[Abstract/Free Full Text]

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Submitted March 10, 1999; accepted November 17, 1999.

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