• quantitative PCR;
  • immunocytochemistry;
  • cytokeratin


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have previously developed a quantitative PCR (QPCR) technique for the detection of cytokeratin 19 (CK19) transcripts in blood and bone marrow and compared this to immunocytochemistry (ICC). Together, both have shown promise for monitoring therapeutic efficacy in patients with metastatic breast cancer. The aim of this study was to determine the feasibility and value of these assays for minimal residual disease (MRD) in monitoring efficacy of adjuvant therapy following surgery for primary breast cancer. Bone marrow aspirates and peripheral blood samples were taken at the time of surgery from patients with primary breast cancer and no evidence of metastases on conventional scans. These were tested for the presence of CK19 mRNA transcripts and cytokeratin positive cells. Follow-up bone marrow aspirates were taken at 3, 6, 12, 24, 36 and 48 months. Prior to surgery, 51% of patients displayed evidence of disseminated cancer cells in the bone marrow by either or both QPCR and ICC. Of 91 patients who had repeat samples assayed, 87% and 65% had positive results at some time using QPCR and ICC, respectively. All patients received adjuvant systemic therapy and in 44 cases where there was a positive result in either the pretreatment or 3-month aspirate, 32/44 (73%) showed a fall in CK19:ABL ratio (QPCR) and 15/24 (63%) showed a reduction in the number of cytokeratin-positive cells (ICC) during follow-up. These results indicate that MRD persists despite adjuvant therapy in a majority of patients with primary breast cancer up to 4 years following surgery. © 2004 Wiley-Liss, Inc.

Breast cancer is unpredictable in its pattern and time of distant relapse. While in some patients the disease recurs soon after removal of the primary tumor, patients continue to relapse over prolonged periods. This feature, together with the current paucity of markers, both biochemical and immunocytochemical, poses significant problems for the physician in determining the optimal form and duration of adjuvant therapy. Currently, conventional preoperative staging investigations including chest radiography, liver ultrasound and skeletal scintigraphy identify occult metastases in approximately 1% of patients1 and have been abandoned in many centers unless clinically indicated.

Greater prognostic information is gained from clinicopathologic staging. Tumor size, grade, the presence of lymphovascular invasion, axillary lymph node involvement and steroid receptor status are important prognostic indicators.2 Despite this, many patients with favorable prognostic features will relapse within 5 years and many patients with poor prognostic features survive for more than 10 years. Studies from our unit and others have shown that many patients with primary breast cancer have disseminated cancer cells.3, 4, 5, 6

The presence of disseminated cancer cells as detected by polymerase chain reaction (PCR) and immunocytochemistry (ICC) in stage I and II breast cancer patients correlates with other prognostic indicators for early relapse such as tumor size, grade, lymphovascular invasion, local lymph node infiltration and with overall survival.4, 5, 6, 7, 8, 9, 10 These different studies using either PCR or ICC to detect a number of different antigens at the time of surgery4, 5, 6, 10, 11 showed a positive detection rate ranging from 25% to 43% depending on the marker used and the number of sites aspirated.

It has been suggested that the monitoring of minimal residual disease (MRD), as defined by the presence of disseminated cancer cells, could be used to improve disease staging, as a marker for evaluating new therapeutic strategies and assessing treatment response in individual patients. Previous studies have shown a very low incidence of MRD in the peripheral blood of primary breast cancer patients with early-stage disease.12, 13, 14

There is considerable variation in the incidence of MRD in both primary and metastatic breast cancer patients. The bone marrow is positive for disseminated cancer cells in 25–50% of patients using PCR14 and 20–40% using ICC.4, 5, 7, 8, 9, 10 Although our earlier study showed a lower incidence of micrometastases immediately after surgery,15 another group obtained a further sample after chemotherapy and showed that there was still evidence of MRD.16

Cytokeratin-19 (CK19) has been used as a marker for disseminated epithelial cancer cells in a number of studies in different solid tumors.12, 13, 14, 17, 18, 19 Other markers such as mammaglobin20, 21, 22, 23, 24, 25, 26 and maspin27, 28, 29, 30, 31 have recently been put forward as breast-specific markers for disseminated malignant cells. However, to date, none has been fully evaluated clinically.

We have developed a quantitative reverse transcriptase-PCR (RT-PCR) method for CK19 measurement (QPCR).32, 33 In this, the number of CK19 transcripts is expressed as a ratio as compared to the number of transcripts from the Abelson oncogene (ABL),34, 35 enabling us to distinguish benign from neoplastic samples. Our previous study used both QPCR and ICC on 145 peripheral blood samples from 22 patients with known metastatic breast cancer.33 There was a significant correlation (p < 0.0001) between the numbers of cytokeratin-positive cells by ICC with CK19 QPCR. There was also a correlation between the 2 assays and response indicating that circulating tumor cell levels reflect changes in disease load. This observation suggested that it might be possible to use QPCR and ICC to monitor residual disease at follow-up and to evaluate any changes with systemic treatment in patients following treatment of primary breast cancer.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References


Consecutive patients diagnosed with early-stage breast cancer were invited to take part in the study. All women were attending Charing Cross Hospital, Queen Mary's Hospital, or West Middlesex Hospital (London, U.K.) between September 1997 and March 2002. All patients had cytologically confirmed primary breast cancer and no evidence of distant metastases on chest radiology, bone scanning, or liver ultrasound (one patient with an abnormal bone scan has been excluded from all analyses). The study was approved by each local ethics committee and was conducted in accordance with the Declaration of Helsinki and all patients gave written informed consent. Samples were blinded for analysis and patients understood that the results would not be made available to them.

The initial bone marrow sample was taken together with peripheral blood at the time of surgery under general anesthetic from both the right and the left posterior iliac crest. Follow-up samples were taken at 3, 6, 12, 24, 36 and 48 months after surgery under local anesthetic and taken from one side only. The iliac crest for sampling at the first follow-up was determined by the QPCR results of the initial sample and the side with the highest CK19:ABL ratio32, 33 was used. At the subsequent follow-up, the QPCR result of the previous sample determined the side to be sampled. If the initial sample was negative on both sides, the follow-up sample was obtained from either the left or the right iliac crest. If, however, the sample became positive, then subsequent samples were obtained from the same side.


The majority of patients underwent wide local excision (81%) and had clinical T136 disease (70%). Pathologic nodal status was assessed in 76% of patients, of whom 58% were N0 (Table I). All patients received adjuvant systemic therapy. Of the 31 (24%) patients who received chemotherapy, 21 received FEC:5-fluorouracil 600 mg/m,2 epirubicin 50 mg/m2 (or 75 mg/m2) and cyclophosphamide 600 mg/m2 3–4 weekly for 6 months. Four patients received CMF:cyclophosphamide 600 mg/m,2 methotrexate 40 mg/m2 and 5-fluorouracil 600 mg/m2 on days 1 and 8 of a 4 weekly cycle for 6 months; 6 patients received epirubicin as 50 mg/m2 on days 1 and 8 of a 4 weekly cycle for 6 months. Eighty-six patients (66%) with estrogen receptor-positive tumors received hormonal therapy (tamoxifen 20 mg/day for all except one patient who received prostap 3.75 mg/month) alone. Sixteen of the patients who received tamoxifen received it in combination with chemotherapy, 15 received chemotherapy alone and 14 received no therapy.

Table I. Clinical Baseline Characteristics of the 131 Patients Recruited
 Wide local excision10681
 Simple mastectomy1411
 Invasive ductal9875
 Invasive lobular2015
Nodal status  
 Not assessed3224

Preparation of bone marrow samples

The skin was incised before the aspirates were taken to minimize the risk of epithelial contamination. Between 2 and 5 ml of bone marrow was aspirated from each side using disposable 15 G (1.8 mm) marrow gauge bone marrow needles (Rocket Medical, Watford, U.K.), into syringes primed with preservative-free heparin (Leo Labs, Risborough, U.K.).

Blood and bone marrow samples were prepared as previously described.32, 33 Briefly, the mononucleocytes were separated from the blood over Ficoll (Amersham, Buckinghamshire, U.K.). The interface cells were removed, washed and the red cells lysed followed by a repeated wash. The mononuclear cells were counted and aliquoted for QPCR and ICC on the basis of ideally 6 × 106 cells for each methodology but with a minimum of 3 × 106 cells for each. Those undertaking the QPCR and ICC were blind to the clinical status and the identity of the patients and their previous assay results.


Cells were cytocentrifuged at a concentration of 5 × 105 per area, air-dried and stained as previously described.32, 33, 37 A total of 6 areas were stained; 4 were stained for the presence of cytokeratin positive cells and 2 were negative controls. The primary antibody (A45-B/B3; Micromet, Munich, Germany) directed to a common epitope of cytokeratin38 was used at 2 mg/ml. The rabbit antimouse antiserum (Z259; Dako, Hamburg, Germany) and the alkaline phosphatase antialkaline phosphatase (APAAP) complex (D651; Dako) were used as recommended by the manufacturer and the reaction developed with new fuschin. An isotype IgG1 mouse myeloma antibody MOPC-21 (Sigma, St. Louis, MO) served as negative control and the MCF-7 cell line as a positive control.32, 33, 37 The cytospins were counterstained with hematoxylin and screened using the Automated Cellular Imaging System (ACIS; Chromavision, San Juan Capistrano, CA). Samples that were isotype-positive were deemed uninterpretable and therefore excluded from the results. During this study, we have screened a large number of bone marrow aspirates from subjects without breast cancer and found them to be all negative.


Synthesis of cDNA was performed as described previously.32, 33 Samples were tested initially for CK19 by nested PCR followed by quantitation for all positive samples as previously described.32, 33

All pre-PCR manipulations were performed in a PCR-only designated laminar flow cabinet and using plugged pipette tips. At least 2 negative controls were included per run and prepared last. The reaction products were electrophoresed on a 1.8% agarose gel in a separate room using dedicated pipettes. A band of 463 bp was visualized for CK19-positive samples. Ethidium bromide-stained gels were used, as Southern blotting would only have served to increase the sensitivity of both methodologies thus resulting in the same ratio.

Quantitative PCR

Competitive QPCR was performed as previously described.32, 33, 34 Briefly, 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 first-step mix. The competitor PCR product was seen at 588 bp and equivalence points were estimated by inspection, which has a low variation (17%) with an experienced operative.34 This is comparable to the variation seen in assays using TaqMan technology where intraassay variation ranges from 11% to 24%.39, 40, 41

Quantification of ABL transcripts as an internal control for the amount and quality of cDNA32, 33, 34 was performed for all samples by a single-step QPCR. Bands were visualized at 385 bp for ABL and 486 bp for the competitor. QPCR results were expressed as the ratio of CK19:ABL. A CK19:ABL ratio greater or equal to 0.1% (1 CK19/1,000 ABL transcripts) was regarded as positive. Conversely, samples with a ratio less than 0.1% were deemed negative. In our previous studies, we have screened 43 normal bone marrows32, 33 and never observed levels in excess of this.

Statistical analyses

The results presented here are preliminary and as such are primarily descriptive. Recruitment to the cohort has stopped but follow-up of patients continues. Chi-square-based tests were used to assess associations between baseline characteristics and MRD positivity at surgery. Positivity rates at each time point are reported and trends over time of the number of patients positive by QPCR, ICC, either technique and both are presented graphically for all data and the subgroup of patients with 12-month follow-up. Matched pairwise comparisons of CK19:ABL ratio values used the Wilcoxon signed-rank test.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients and baseline MRD results

One hundred thirty-one patients were recruited into the study with a mean age of 58 years (range, 31–84 years). Of these, 93 (71%) agreed to continue after surgery and 74 have been followed up after 3 months (Table II). Of these, 60 subjects have been followed up for 48 months but only 31 (35%) have given bone marrow samples at 6, 12, 24, 36 and 48 months. Four patients have relapsed; of these, 2 have subsequently died.

Table II. Summary of Results of MRD Detection of All Patients in the Study by ICC and QPCR
Bone marrow sampleNumber of patientsPositivity
  1. Summary of results of detection of CK19-positive cells in bone marrow aspirates at surgery to 48 months postsurgery (QPCR positivity of > 0.1% CK19:ABL). The combined column indicates the results of both aspirates at the time of surgery by ICC and/or QPCR and of combining ICC and/or QPCR positivity at follow-up (see also Fig. 1).

Surgery13134/109 (31%)54/131 (41%)68/131 (51%)
3 months8814/77 (18%)26/88 (30%)34/88 (39%)
6 months7411/69 (16%)13/74 (18%)21/74 (28%)
12 months6921/66 (32%)32/69 (46%)41/69 (59%)
24 months6515/46 (33%)16/65 (25%)24/65 (37%)
36 months439/38 (24%)14/43 (33%)16/43 (37%)
48 months319/31 (29%)8/31 (26%)11/31 (35%)

As this is the first attempt to use QPCR in the detection/monitoring of MRD in primary breast cancer patients, QPCR and ICC (which has been widely used for detection of MRD) were compared in the 558 samples where both results were available (110 patients at surgery-left plus 109 at surgery-right plus all ICC at follow-up; Table II). Although in the majority of cases (71%), the 2 methodologies were in agreement, this was principally because of the concordance of dual negative findings. Where a sample was found to be positive, disagreement between the 2 tests was predominantly where a positive result was seen by QPCR but the ICC result was negative. This is illustrated in Table III, which details results of 51 patients with complete data to 12 months (i.e., no missing data points or isotype-positive ICC). There were no significant relationships between the baseline characteristics of the patients (Table I) and a positive finding by ICC or QPCR.

Table III. Results of MRD Detection in Bone Marrow Aspirates from All Patients up to 12 Months
 Baseline (left)Baseline (right)3 months6 months12 months
  • Summary of results of detection of CK19-positive cells in bone marrow aspirates from (all patients with complete data from surgery to 12 months postsurgery (n = 51). QPCR data are the ratio of CK19:ABL expressed as a percentage.

  • 1

    The presence of 1–2 cytokeratin-positive cells.

  • 2

    Three or more cytokeratin-positive cells.

  • 3

    > 0.1 is a positive result.

10 010 0.063 0.025 
2020.079 0.0316 0.077 0.031
30 0.063 010.13 0.0311
40 0 0.1593 0.04 0.0251
5010.25310 0.02 0 
60.016 0 0.1253 0 0.016 
70.063 0 0.03110.03120.2531
80 0 0 020.0771
90 0 0.02 0.13 0.077 
1001010 0 0 
110.1253 1.253 0.125310 0.79432
120.05 0 0.031 0.02510.0771
130 0 0 010.1253 
140.077 0 0 0.016 01
150.07710 0.07710.025 01
160.13 0.05 0.0253 0.0510.0771
170 0 0 0.05 0.13 
180.077 0.167310.25 0.05 0.431
1902010.04 0 0 
200.063 0.13 0.02 0.04 0.431
21010.13 0.016 0.05 0.131
220.077 0.05 0.013 010.051
230.06310.077 0.253 0.253 0.15932
240 0 0.05 0.077 0.13 
250.04 0 0 0.031 0.313 
260.063 0.13 0 0.077 0.04 
270.253 0.23 0 0.077 0.132
280.077 0.031 0.077 0.02 0.1593 
290.025 0.025 0.43 0 0.04 
300 0.13 0.1253 0.05 0.1253 
31010.04 0.031 0.025 0.1253 
320 0.04 0.1310.031 0.042
330.13 0.43 0 0 0 
340 0 0.6333 0.077 0.1253
350.025 0.1310.031 0 0.01 
360.077 0.02 0.253 0.077 0.1593 
370.13 0.1593 0.1673 0.05 0.12531
380.031 0 0.025 0.02 0.23 
390.04 0.13 0.05 0.077 0.13 
400 0.05 020.23 0.01 
410.4310.05 0 0.05 0.13 
4213 0.53 1 0.13 0.1253 
430.077 0.159320.05 0 0.031 
440.05 0.13 0.04 0.05 0.077 
450 0 0.6253 0.0625 0.01593 
460 0.0520.1310.02 0.01593 
470 0 01020.43 
480.05 0.05 020.077 0.253 
490.13 0.1593 0.016 0 0.04 
500.063 0.13 0 0.253 1.88731
510.2310.159320 0 0.071 

At surgery, 68/131 (51%) patients had positive results using either or both methodologies (Table II, Fig. 1a). The number of positive patients as determined by ICC (31%) was consistent with previously published studies using ICC at surgery.4, 5, 6, 10, 11

thumbnail image

Figure 1. (a) Graph showing the trends in CK19 positivity by QPCR (filled circle), immunocytochemistry (filled square) and either QPCR or ICC (filled triangle) and both ICC and QPCR (open circle). (b) CK19:ABL ratio over a period of 48 months.

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We carried out a separate analysis for those samples that were positive using both techniques (i.e., double positive). Of all samples taken, 63/558 (11%) were double positive either at surgery or on follow-up. Only 21 of 109 (19%) patients were double positive at surgery (Fig. 1, both ICC and QPCR). However, these patients did not differ in terms of their clinicopathologic characteristics from the patients who had only one or other test positive at surgery. Only 31 of 93 subjects had a double positive aspirates during follow-up, but again, there was no significant difference between these and other patients in terms of conventional prognostic indexes. Of the 165/558 (30%) samples positive by QPCR, 63 (38%) were also positive by ICC. Of the 124/558 (22%) samples positive by ICC, 63 (51%) were also positive by QPCR. This demonstrates the higher sensitivity of the QPCR as compared to the ICC in terms of samples positive and the discrepancies may be due to the 2 techniques measuring different markers and due to sampling problems arising from analyzing a single bone marrow aspirate by both ICC and QPCR.

The number of positive cells detected by ICC in the aspirates ranged from 1 to 12 cells (except for one patient who had 2 results of 51 and 148 after surgery), but for the vast majority of samples that were positive, 77% had 1 to 2 detectable cells.

Follow-up of all study patients

In contrast to the initial sample at surgery when bilateral aspirates were taken, a single follow-up aspirate was taken during follow-up. At 3 months, 14/77 (18%) were positive by ICC and 26/88 (30%) by QPCR, approximately the same as at surgery if one considers either the left or right aspirate alone. At 6 months after surgery, 11/69 (16%) were positive by ICC and 13/74 (18%) by QPCR. The decrease from surgery to 6 months was significant by QPCR (p = 0.001) but not by ICC (p = 0.09).

At 12 months after surgery, there appeared to be an increase in the proportion of positive patients. This increase, 16% to 32% (ICC) and 18% to 46% (QPCR) from 6 to 12 months, was of statistical significance (p = 0.008 for ICC and p = 0.001 for QPCR). This was initially thought to be an indication of the emergence of resistance to adjuvant therapy; however, the trend was not maintained beyond 12 months. There was no relationship between the characteristics of the breast carcinoma (node status, ERα status, etc.) and whether patient's bone marrows were positive by 12 months with either ICC or QPCR.

At 24 months, the overall proportion of positive patients fell to the 3-month level. Thereafter, the percentage of patients positive for disseminated cells stabilized at approximately 40% for the next 2–3 years if the 2 methodologies are combined (Fig. 1a), indicating that MRD persisted in a substantial proportion of patients for up to (and at least) 48 months. Figure 1(b) shows CK19:ABL ratios for all patients at different time points to illustrate the median and range of values.

Effect of adjuvant therapy

The individual pattern of positivity within a patient was examined to determine whether individual patients showed either rises or falls in MRD during adjuvant therapy. Of the 110 patients with at least 1 ICC sample, 65 (59%) had a positive result at some time and 56/80 (70%) of patients with at least 2 follow-up ICC samples had a positive result. All patients received adjuvant therapy and, in cases where there was a positive result in either the pretreatment or 3-month aspirate and some follow-up data, 32/45 (71%) showed a fall in the CK19:ABL ratio [in 11/32 (34%), this decrease was a single log; in the remaining 21 patients, there was a larger decrease] and 15/30 (50%) showed a reduction in cytokeratin-positive cells detected using ICC during follow-up.

Of interest, in one patient, 977/2 × 106 stained cells were detected in the bone marrow sample taken at surgery and the ratio of CK19:ABL in this patient was similarly very high at 1:2 (50%). Despite this, she had no sign of overt metastatic disease on routine bone scanning, commenced treatment with tamoxifen and was monitored in the same way as the other patients. One year after starting adjuvant therapy, the ratio of CK19:ABL fell to 1.6% and the number of ICC-detectable cells fell to 146/2 × 106 stained cells. This patient was excluded from the statistical analysis because she clearly had extensive metastatic breast cancer confined to the bone marrow.

Results in 51 patients with complete data (12-month results)

We wished to examine the results in the 51 patients who had a complete set of 12-month bone marrow results. These are detailed in Table III and Figure 2. The pattern of results in these patients is similar to the entire cohort (Fig. 1). Table III shows the QPCR and ICC results in this cohort of 51 patients and Figure 3 shows the frequency of the number of positive samples in these 51 patients. Only one patient was positive throughout by QPCR and none by ICC. The majority of patients had 1 or 2 samples positive: 35 (69%) by QPCR and 31 (61%) by ICC. Nine (18%) patients were negative throughout by QPCR and 18 (35%) by ICC. Of the remaining 51 patients, 2 (4%) had 3 samples positive by ICC and 6 (12%) by QPCR and 1 (2%) patient had all samples positive by QPCR. Twenty-three (45%) of these patients were negative at surgery by both QPCR and ICC. All of these had a positive result in at least one follow-up aspirate, although of these, 11/23 (48%) had only a single positive result by QPCR (Table III). This may be due to sampling problems and the splitting of the aspirate for analysis by both ICC and QPCR.

thumbnail image

Figure 2. Graph showing the trends in CK19 positivity in all patients with complete data from surgery to 12 months postsurgery (n = 51) by QPCR (filled circle), immunocytochemistry (filled square), either QPCR or ICC (filled triangle) and both ICC and QPCR (open circle).

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thumbnail image

Figure 3. Graph showing the frequency of positive bone marrow samples in patients with complete data from surgery to 12-month follow-up sample (n = 51) by QPCR (open square) and ICC (filled square).

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At the outset of the study, an analysis of 20 ml of peripheral blood from each patient at each time point until 12 months after surgery was planned. However, only 28/246 samples (11%) were CK19-positive by QPCR and none of 208 by ICC. This low detection rate is in agreement with previously published studies12, 13, 14, 42 and it was thus decided to discontinue analysis of these peripheral blood samples.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Following surgery for primary breast cancer, approximately 70% of women with early breast cancer now receive tamoxifen or chemotherapy for a fixed time interval but no attempt is made to ascertain whether it is effective in individual patients. Only a proportion of patients benefit and therefore many experience treatment-related side effects for little benefit. If a monitoring system could be developed, alternative therapies could be given to nonresponding patients, with the expectation that more patients would be cured, as prolonged follow-up of trials of adjuvant therapies have demonstrated that a proportion of patients are cured by systemic therapy.43

Bone marrow samples from primary breast cancer patients were analyzed for presence of micrometastases. Bone marrow was aspirated at the time of excision of the primary tumor and at 3, 6, 12, 24, 36 and 48 months after surgery. There were major losses to follow-up between surgery and 3 months (some patients were unwilling to have aspirates taken so close to surgery) and from 3 to 6 months (some patients were unwilling to return after the first aspiration carried out under local anesthetic). Withdrawals after 6 months were less frequent and collection of follow-up samples continues. Annual bone marrow aspirates appear acceptable to the majority of patients.

Our results indicate that a variable pattern of MRD changes is observed after surgery and adjuvant treatment for breast cancer. However, a significant proportion of patients shows a decline in the number of cytokeratin transcripts following surgery and adjuvant therapy. We speculate that the significant decrease in the proportion of patients positive by QPCR at 6 months is probably due to the surgical removal of the primary tumor together with the effects of adjuvant therapy. There is evidence in animal studies that tumor removal may stimulate cell proliferation in macrometastatic foci44, 45 and that removal caused a switch of micrometastatic foci to an angiogenic phenotype, thus resulting in growth of metastases.46 No direct evidence of these data has been demonstrated in human breast cancer; however, one study does show some evidence that removal of the primary tumor may induce changes in the growth kinetics of metastatic foci.47 To investigate the rise in the proportion of patients positive at 12 months and the subsequent fall, we considered the relationship between CK19 positivity and adjuvant treatment (chemotherapy alone or in combination with hormonal therapy). Although there were only a limited number of chemotherapy-treated patients in this cohort (n = 9), no treatment-specific associations were observed (data not shown).

Some patients appear to show a rise in MRD despite therapy, but it is too early to judge the significance of this observation, since only 4 patients in this series have clinically confirmed relapses (2/4 were positive at surgery by QPCR and 1/4 by ICC).

Many groups have examined sequential changes in biochemical markers. We have previously measured 12 different markers from diagnosis to relapse, but only demonstrated a lead interval (as defined by the time between the detection of markers of micrometastases and the appearance of clinically or radiologically overt metastases) in 3 markers: carcinoembryonic antigen (CEA), serum alkaline phosphatase (SAP) and γ-glutamyl-transpeptidase (GGT); for these 3, the mean lead interval was approximately 3-month duration. This result was confirmed recently by Sutterlin et al.,48 who measured CEA and carbohydrate antigen 15-3 (CA 15-3) for their predictive value. The diagnostic accuracy was only 83% for CEA and 88% for CA15-3. CEA and CA15-3 had a positive predictive value of 27% and 47%, respectively. Molina et al.49 recorded lead times of 2–5 months with circulating c-erb B2, CEA and CA15-3. Other studies have focused on defining possible markers for monitoring metastatic breast cancer; these studies have included CA15-3 and the erythrocyte sedimentation rate (ESR),50 CA15-3, CEA and TPS.51 These studies show that the rate of fall of tumor markers relate to outcome. However, to date, considerable controversy surrounds the value of monitoring patients using these markers. It has been proposed that as disseminated cells are highly resistant to chemotharapy16 and that the presence of tumor cells in bone marrow after high-dose chemotherapy is not associated with a poor prognosis,52 merely monitoring the presence of disseminated tumor cells is insufficient to predict relapse. Studies on the biology of the disseminated tumor cells may improve the prediction of relapse, in particular the proliferative potential of the cells.53, 54 The proliferative potential was demonstrated to be a better indicator of decreased patient survival than ICC; however, neither of these studies were used for monitoring follow-up samples. Therefore, this methodology requires further investigation.

Real-time PCR is now becoming the standard methodology for quantifying gene transcripts and, using Lightcycler Technology (Roche Diagnostics, Mannheim, Germany), we have developed QPCR assays for both CK19 and ABL using the artificial competitor we made previously32 in order to construct an internal standard curve for absolute quantification of CK19 and ABL transcripts (data not shown). The nested QPCR had an intraassay variability of 6–13% when 2 samples (1 borderline positive, 1 highly positive) were assayed 6 times in the same run and the interassay variability (same sample assayed 6 times on consecutive runs) was 10.5%. When the 2 QPCR methodologies were compared in 49 patient samples, there was an extremely good correlation between the 2 (p = 0.0002). Real-time PCR shows increased sensitivity and reproducibility of the test for further studies. A recent study looking at CK19 transcripts in peripheral blood of breast cancer patients55 has employed a similar strategy. With the development of real-time PCR, this may become the standard methodology for monitoring breast cancer patients. A further possibility for optimization is the development of automated imaging systems. Several are now commercially available; however, only further research will determine whether this is indeed the case. We recommend using both methodologies in the detection of bone marrow micrometastases because of the uncertainty inherent in detecting such small numbers of cells by ICC alone in a large sample.

In conclusion, we have demonstrated that in a substantial proportion of patients, MRD persists using the techniques that we have developed, and that it is possible to monitor patients, preferably using both QPCR and ICC, after breast surgery using bone marrow aspirates.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Professor Klaus Pantel (Universitätsklinikum Eppendorf, Hamburg, Germany) for his invaluable technical assistance and advice.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Coombes RC, Powles TJ, Abbott M, De Rivas L, Ford HY, McCready VR, Neville AM, Gazet JC. Physical test for distant metastases in patients with breast cancer. J R Soc Med 1980; 73: 61723.
  • 2
    Fisher B, Dignam J, Bryant J, DeCillis A, Wickerham D, Wolmark N, Costantino J, Redmond C, Fisher E, Bowman D, Deschenes L, Dimitrov N, et al. Five versus more than five years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors. J Natl Cancer Inst 1996; 88: 152942.
  • 3
    Redding WH, Coombes RC, Monaghan P, Clink HM, Imrie SF, Dearnaley DP, Ormerod MG, Sloane JP, Gazet JC, Powles TJ. Detection of micrometastases in patients with primary breast cancer. Lancet 1983; 2: 12714.
  • 4
    Mansi JL, Gogas H, Bliss JM, Gazet JC, Berger U, Coombes RC. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999; 354: 197202.
  • 5
    Diel IJ, Kaufmann M, Costa SD, Holle R, von Minckwitz G, Solomayer EF, Kaul S, Bastert G. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 16528.
  • 6
    Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CRM, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000; 342: 52533.
  • 7
    Pantel K, Cote RJ, Fodstad O. Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999; 91: 111324.
  • 8
    Diel IJ, Kaufmann M, Goerner R, Costa SD, Kaul S, Bastert G. Detection of tumor cells in bone marrow of patients with primary breast cancer: a prognostic factor for distant metastasis. J Clin Oncol 1992; 10: 15349.
  • 9
    Braun S, Cevatli BS, Assemi C, Janni W, Kentenich CRM, Schindlbeck C, Rjosk D, Hepp F. Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy. J Clin Oncol 2001; 19: 146875.
  • 10
    Harbeck N, Untch M, Pache L, Eiermann W. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3-year median follow-up. Br J Cancer 1994; 69: 56671.
  • 11
    Cote RJ, Rosen PP, Lesser ML, Old LJ, Osborne MP. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991; 9: 174956.
  • 12
    Datta YH, Adams PT, Drobyski WR, Ethier SP, Terry VH, Roth MS. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994; 12: 47582.
  • 13
    Aihara T, Noguchi S, Ishikawa O, Furukawa H, Hiratsuka M, Ohigashi H, Nakamori S, Monden M, Imaoka S. Detection of pancreatic and gastric cancer cells in peripheral and portal blood by amplification of keratin 19 mRNA with reverse transcriptase-polymerase chain reaction. Int J Cancer 1997; 72: 40811.
  • 14
    Schoenfeld A, Luqmani Y, Smith D, O'Reilly S, Shousha S, Sinnett HD, Coombes RC. The detection of micrometastases in the lymph nodes, peripheral blood and bone marrow of patients with breast cancer using immunocytochemistry and polymerase chain reaction. In: Breast cancer: advances in biology and therapeutics, (John Libbey Eurotext, Paris). 1996. 289302.
  • 15
    Mansi JL, Berger U, McDonnell T, Pople A, Rayter Z, Gazet JC, Coombes RC. The fate of bone marrow micrometastases in patients with primary breast cancer. J Clin Oncol 1989; 7: 4459.
  • 16
    Braun S, Kentenich C, Janni W, Hepp F, de Waal J, Willgeroth F, Sommer H, Pantel K. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000; 18: 806.
  • 17
    Schoenfeld A, Luqmani Y, Smith D, O'Reilly S, Shousha S, Sinnett HD, Coombes RC. Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction. Cancer Res 1994; 54: 298690.
  • 18
    Van Trappen PO, Gyselman VG, Lowe DG, Ryan A, Oram DH, Bosze P, Weekes AR, Shepherd JH, Dorudi S, Bustin SA, Jacobs IJ. Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet 2001; 357: 1520.
  • 19
    Schoenfeld A, Luqmani Y, Sinnett HD, Shousha S, Coombes RC. Keratin 19 mRNA measurement to detect micrometastases in lymph nodes in breast cancer patients. Br J Cancer 1996; 74: 163942.
  • 20
    Aihara T, Fujiwara Y, Ooka M, Sakita I, Tamaki Y, Monden M. Mammaglobin B as a novel marker for detection of breast cancer micrometastases in axillary lymph nodes by reverse transcription-polymerase chain reaction. Breast Cancer Res Treat 1999; 58: 13740.
  • 21
    Grunewald K, Haun M, Urbanek M, Fiegl M, Muller-Holzner E, Gunsilius E, Dunser M, Marth C, Gastl G. Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19. Lab Invest 2000; 80: 10717.
  • 22
    Leygue E, Snell L, Dotzlaw H, Hole K, Troup S, Hiller-Hitchcock T, Murphy LC, Watson PH. Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol 1999; 189: 2833.
  • 23
    Marchetti A, Buttitta F, Bertacca G, Zavaglia K, Bevilacqua G, Angelucci D, Viacava P, Naccarato A, Bonadio A, Barassi F, Felicioni L, Salvatore S, Mucilli F. mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients. J Pathol 2001; 195: 18690.
  • 24
    O'Brien N, Maguire TM, O'Donovan N, Lynch N, Hill AD, McDermott E, O'Higgins N, Duffy MJ. Mammaglobin a: a promising marker for breast cancer. Clin Chem 2002; 48: 13624.
  • 25
    Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996; 56: 8605.
  • 26
    Zehentner BK, Dillon DC, Jiang Y, Xu J, Bennington A, Molesh DA, Zhang X, Reed SG, Persing D, Houghton RL. Application of a multigene reverse transcription-PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes. Clin Chem 2002; 48: 122531.
  • 27
    Sabbatini R, Federico M, Morselli M, Depenni R, Cagossi K, Luppi M, Torelli G, Silingardi V. Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. J Clin Oncol 2000; 18: 191420.
  • 28
    Merrie AE, Yun K, Gunn J, Phillips LV, McCall JL. Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer. Br J Cancer 1999; 80: 201924.
  • 29
    Lopez-Guerrero JA, Gilabert PB, Gonzalez EB, Sanz Alonso MA, Perez JP, Talens AS, Oraval EA, de la Rubia Comos J, Boix SB. Use of reverse-transcriptase polymerase chain reaction (RT-PCR) for carcinoembryonic antigen, cytokeratin 19, and maspin in the detection of tumor cells in leukapheresis products from patients with breast cancer: comparison with immunocytochemistry. J Hematother 1999; 8: 5361.
  • 30
    Leone F, Perissinotto E, Viale A, Cavalloni G, Taraglio S, Capaldi A, Piacibello W, Torchio B, Aglietta M. Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction. Bone Marrow Transplant 2001; 27: 51723.
  • 31
    Corradini P, Voena C, Astolfi M, Delloro S, Pilotti S, Arrigoni G, Bregni M, Pileri A, Gianni AM. Maspin and mammaglobin genes are specific markers for RT-PCR detection of minimal residual disease in patients with breast cancer. Ann Oncol 2001; 12: 16938.
  • 32
    Slade MJ, Smith BM, Sinnett HD, Cross NC, Coombes RC. Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 1999; 17: 8709.
  • 33
    Smith BM, Slade MJ, English J, Graham H, Luchtenborg M, Sinnett HD, Cross NC, Coombes RC. Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol 2000; 18: 14329.
  • 34
    Cross NCP, Lin F, Chase A, Bungey J, Hughes TP, Goldman JM. Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 1993; 82: 192936.
  • 35
    Cross NCP, Hughes TP, Lin F, O'Shea P, Bungey J, Marks DI, Ferrant A, Martiat P, Goldman JM. Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlation with acute graft-versus-host disease and relapse. Br J Haematol 1993; 84: 6774.
  • 36
    Yarbro JW, Page DL, Fielding LP, Partridge EE, Murphy GP. American Joint Committee on Cancer prognostic factors consensus conference. Cancer 1999; 86: 243646.
  • 37
    Pantel K, Schlimok G, Angstwurm M, Weckermann D, Schmaus W, Gath H, Passlick B, Izbicki JR, Riethmuller G. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor markers in bone marrow. J Hematother 1994; 3: 16573.
  • 38
    Stigbrand T, Andres C, Bellanger L, Bishr Omary M, Bodenmuller H, Bonfrer H, Brundell J, Einarsson R, Erlandsson A, Johansson A, Leca JF, Levi M, et al. Epitope specificity of 30 monoclonal antibodies against cytokeratin antigens: the ISOBM TD5-1 workshop. Tumour Biol 1998; 19: 13252.
  • 39
    Anhuf D, Eggermann T, Rudnik-Schoneborn S, Zerres K. Determination of SMN1 and SMN2 copy number using TaqMan technology. Hum Mutat 2003; 22: 748.
  • 40
    Dehee A, Asselot C, Piolot T, Jacomet C, Rozenbaum W, Vidaud M, Garbarg-Chenon A, Nicolas JC. Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR. J Med Virol 2001; 65: 54352.
  • 41
    Yun Z, Lewensohn-Fuchs I, Ljungman P, Ringholm L, Jonsson J, Albert J. A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients. J Virol Methods 2003; 110: 739.
  • 42
    Schoenfeld A, Kruger KH, Gomm J, Sinnett HD, Gazet JC, Sacks N, Bender HG, Luqmani Y, Coombes RC. The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 1997; 33: 85461.
  • 43
    Early Breast Cancer Trialists' Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998; 351: 145167.
  • 44
    Fisher B, Gunduz N, Coyle J, Rudock C, Saffer E. Presence of a growth-stimulating factor in serum following primary tumor removal in mice. Cancer Res 1989; 49: 19962001.
  • 45
    Gunduz N, Fisher B, Saffer EA. Effect of surgical removal on the growth and kinetics of residual tumor. Cancer Res 1979; 39: 38615.
  • 46
    Holmgren L, O'Reilly MS, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995; 1: 14953.
  • 47
    Demicheli R, Valagussa P, Bonadonna G. Does surgery modify growth kinetics of breast cancer micrometastases? Br J Cancer 2001; 85: 4902.
  • 48
    Sutterlin M, Bussen S, Trott S, Caffier H. Predictive value of CEA and CA 15-3 in the follow up of invasive breast cancer. Anticancer Res 1999; 19: 256770.
  • 49
    Molina R, Jo J, Filella X, Zanon G, Farrus B, Munoz M, Latre M, Pahisa J, Velasco M, Fernandez P, Estape J, Ballesta A. C-erbB-2, CEA and CA 15.3 serum levels in the early diagnosis of recurrence of breast cancer patients. Anticancer Res 1999; 19: 25515.
  • 50
    Robertson J, Jaeger W, Syzmendera J, Selby C, Coleman R, Howell A, Winstanley J, Jonssen P, Bombardieri E, Sainsbury J, Gronberg H, Kumpulainen E, Blamey R. The objective measurement of remission and progression in metastaticbreast cancer by use of serum tumour markers: European Group for Serum Tumour Markers in Breast Cancer. Eur J Cancer 1999; 35: 4753.
  • 51
    van Dalen A, van der Linde D, Heering K, van Oudalblas A. How can treatment response be measured in breast cancer patients? Anticancer Res 1993; 13: 19014.
  • 52
    Kruger WH, Kroger N, Togel F, Renges H, Badbaran A, Hornung R, Jung R, Gutensohn K, Gieseking F, Janicke F, Zander AR. Disseminated breast cancer cells prior to and after high-dose therapy. J Hematother Stem Cell Res 2001; 10: 6819.
  • 53
    Solakoglu O, Maierhofer C, Lahr G, Breit E, Scheunemann P, Heumos I, Pichlmeier U, Schlimok G, Oberneder R, Kollermann MW, Kollermann J, Speicher MR, Pantel K. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci USA 2002; 99: 224651.
  • 54
    Pierga JY, Bonneton C, Magdelenat H, Vincent-Salomon A, Nos C, Pouillart P, Thiery JP. Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer. Br J Cancer 2003; 89: 53945.
  • 55
    Stathopoulou A, Gizi A, Perraki M, Apostolaki S, Malamos N, Mavroudis D, Georgoulias V, Lianidou ES. Real-time quantification of CK19 mRNA-positive cells in peripheral blood of breast cancer patients using the lightcycler system. Clin Cancer Res 2003; 9: 514551.