Japanese Journal of Clinical Oncology 33:167-172 (2003)
© 2003 Foundation for Promotion of Cancer Research
Clinical Significance of Bone Marrow Micrometastasis Detected by Nested RT-PCR for Keratin-19 in Breast Cancer Patients
Departments of 1 Surgery, 2 Pathology and 3 Hemato-oncology, Ajou University School of Medicine, Suwon, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Purpose: Breast cancers in the early phase frequently undergo distant metastasis and survival of patients is greatly dependent on distant metastasis. The occurrence of micrometastasis has been suggested to relate with prognostic features of breast cancer, such as lymph node metastasis and the presence of vascular invasion. The aim of this study was to examine the presence of keratin-19 mRNA of epithelial tumors in bone marrow aspirates obtained from breast cancer patients and its possible correlation with tumor staging and disease-free survival.
Methods: Bone marrow samples were obtained from 59 breast cancer patients at the time of surgery. We separated the mononuclear fraction from the samples and carried out nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of keratin-19 mRNA with two different pairs of primers. After operation, the patients were followed up at 3-month intervals. We studied the possible correlation of the detection of keratin-19 mRNA with tumor size, nodal involvement, stage and recurrence rate.
Results: Bone marrow micrometastasis was detected by nested RT-PCR for keratin-19 mRNA in one of four patients with ductal carcinoma in situ (DCIS), 13 of 30 patients with T1, 11 of 20 patients with T2 and all four patients with T3 lesion. Recurrence was observed in seven cases and all of them were positive for micrometastasis in bone marrow.
Conclusion: The method of nested RT-PCR to detect the presence of keratin-19 mRNA in bone marrow from patients with breast cancer is sensitive and reliable. Moreover, early recurrence was observed in the patients with the tumor mRNA detected in bone marrow. Additional studies with larger numbers of patients and longer follow-up are desirable.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Early distant metastasis is fairly common in breast cancers and recurrence and survival are greatly influenced by the distant metastasis. To minimize recurrence in distant sites, chemotherapy and hormone therapy are commonly used. Major prognostic factors are nodal status, tumor size, histological grade and hormonal receptor status. Theoretically, distant metastasis or recurrence is the result of tumor growth from micrometastasis already existing at the time of surgery. Therefore, many efforts have been made to uncover micrometastasis in distant organs to characterize the cancer biologically and to determine subclinical staging. Thus far, two methods have been used for this purpose: one of them is the immunochemical staining of the nucleated cells from bone marrow with epithelial markers and the other is the detection of epithelial mRNA markers from the marrow cells with the reverse transcriptase polymerase chain reaction (RT-PCR) technique (1,2). We report here the results of our prospective study on clinical significance of bone marrow micrometastasis detected with nested RT-PCR for keratin-19 mRNA in breast cancer patients.
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PATIENTS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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During the period between 1995 and 1998, 303 female patients were diagnosed as having breast cancer and were treated at the Ajou University Hospital, Suwon, Korea. Of these patients, 59 consented to our prospective study and were enrolled. These patients had undergone breast operation and bone marrow aspiration from the iliac spine and were treated and subjected to follow-up studies similarly to other breast cancer patients, regardless of the results of the bone marrow RT-PCR study. The minimum follow-up time was 4 years. The clinical and pathological data and the results of the bone marrow keratin-19 nested RT-PCR were reviewed and analyzed together with clinical results on recurrence and survival.
Bone marrow aspiration was carried out from the iliac spine preoperatively at the time of breast surgery under general anesthesia. Total RNA was extracted from bone marrow, as described in the next section, using guanidine thiocyanate–phenol–chloroform. Keratin-19 mRNA was amplified by RT-PCR using two pairs of primers (Table 1). The final product was separated in a 2% agarose gel containing ethidium bromide. To normalize relative levels of expression, ß-actin was used as an internal reference control.
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RNA Isolation
A 10 ml amount of bone marrow obtained from breast cancer patients was carefully layered over 10 ml of Lymphoprep (Nycomed). Nucleated cells were spun down at 2700 r.p.m. for 20 min and stored at –70°C until used. These cells were dissolved in TRIzol reagent (Gibco BRL) and RNA was isolated according to the manufacturer’s instructions and quantified spectrophotometrically at 260 nm.
Oligonucleotide Primer Design
Four primers were designed to generate a PCR product that spans the translated region of keratin-19 mRNA. The external primer set used in RT-PCR, K19ES (external sense) 5'-aggtggattccgctccggbca-3' and K19EA (external antisense) 5'-atcttcctgtccctcgagc-3', generates a 461 base pair (bp) product. The inner primer set used in nested RT-PCR, K19IS (internal sense) 5'-agtgtgtcttccaaggcagc-3' and K19IA (internal antisense) 5'-gacatgcgaagccaatatgagg-3', produces a fragment of length 221bp (Table 1).
RT-PCR and Nested PCR
A 30 µl RT-PCR reaction volume contained 50 mM KCl, 10 mM Tris–HCl (pH 9.0), 0.1% Triton X-100, 200 µM each of dNTP, 2.5 mM MgCl2, 0.1 mM DTT, 60 U M-MLV reverse transcriptase, 0.5 U of tag polymerase, 12 U of RNase inhibitor, 10 mM external sense primer, 10 mM external antisense primer, 1 µg of total RNA and DEPC-DW. Complementary DNA was made at 42°C for 60 min. The cycle conditions were as follows: 2 min at 95°C, 35 cycles of 1 min at 94°C (denaturation), 1.5 min at 57°C (annealing), 2 min at 72°C (extension) and lastly 10 min at 72°C. A 2 µl volume of the first amplification product was mixed with 18 µl of the second PCS buffer containing 50 mM KCl, 10 mM Tris–HCl (pH 9.0), 0.1% Triton X-100, 200 µM each of dNTP, 2.5 mM MgCl2, 0.5 U of tag polymerase, 10 mM internal sense primer, 10 mM internal antisense primer and DEPC-DW. The reaction cycle was the same as described above. Thermal cycling was performed in a GeneAmp 9600 apparatus (Perkin-Elmer). Each sample was subjected to electrophoresis with 2% agarose gels, stained with ethidium bromide and visualized on a transilluminator. The presence of intact RNA was confirmed by RT-PCR using ß-actin-specific primers (Fig. 1).
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To determine the sensitivity of RT-PCR assays, 107 peripheral blood mononuclear cells obtained from a normal donor were mixed with decreasing numbers of MCF-7 cell lines (for the last dilution, one breast cancer cell was diluted in 107 normal cells). To determine the specificity for keratin-19, bone marrow aspirates obtained from 10 normal women were used as control samples. RNA was isolated and RT-PCR analysis was performed as described above.
Patient Surveillance and Statistics
We evaluated recurrence of the disease by physical examination, serological testing, chest PA, abdominal ultrasonography and bone scans at 3 month intervals.
The Mann–Whitney test was used to evaluate the correlation between bone marrow micrometastasis and other prognostic factors and recurrence. Disease-free survival comparisons between groups with CK-19 positive and negative results were conducted using the Kaplan–Meier test and log-rank test. Disease-free survival comparisons between each stage were performed in the same manner. Values of P < 0.05 were considered statistically significant. Multivariate analysis was not conducted owing to the small patient numbers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 2, the sensitivity of the assay appeared to be highly satisfactory, so that nested RT-PCR could detect 10 breast cancer cells in 10 million nucleated blood cells. In 10 normal bone marrow aspirates, there was one positive case for nested RT-PCR with keratin-19 (Fig. 3).
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Relationship Between the Results of RT-PCR and Tumor Size, Lymph Node Involvement and Stage
As shown in Table 2, among four ductal carcinoma in situ (DCIS) patients, there was one positive case of nested RT-PCR. Theoretically, they should show a negative reaction. There were 13 positive cases among 30 patients with T1, 11 positive cases among 20 patients with T2, all four positive cases with T3 and one negative case with T4. According to node staging, 20 cases were positive among 41 patients with N0, six cases in 11 patients with N1 and three cases in seven patients with N2. According to staging, 11 cases were positive among 26 patients with stage 1, 13 cases in 22 patients with stage 2 and four cases in seven patients with stage 4. However, no statistical significance was found between bone marrow micrometastasis and tumor size, lymph node metastasis and stage.
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Relationship Between the Results of RT-PCR and Recurrence
Table 3 shows that there were seven cases of recurrence among 59 patients. There was no recurrence in four cases with DCIS, one case of recurrence in 30 patients with T1, four cases of recurrence in 20 patients with T2 and two cases of recurrence in four patients with T3.
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According to lymph node metastasis, there were three cases of recurrence in 41 patients with N0, two in 11 patients with N1 and two in seven patients with N2.
There was no recurrence in patients with DCIS and stage 1, four cases of recurrence in 22 patients with stage 2 and three cases of recurrence in seven patients with stage 3.
All seven cases of recurrence had RT-PCR positive for keratin-19 mRNA and there was no recurrence in 30 patients with RT-PCR negative for keratin-19 mRNA.
After follow-up, we found a significant relationship in survival curves between bone marrow micrometastasis and recurrence (P = 0.004) (Fig. 4). Also, there was a significant difference in survival curves between each stage (P = 0.01) (Fig. 5). However, tumor size, lymph node involvement and hormonal receptor did not affect survival curves (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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The most important prognostic factor of breast cancer is lymph node metastasis. Among the biological characteristics of breast cancer, lymph node metastasis is closely related with distant metastasis and long-term survival. In contrast to lymph node metastasis, tumor size is related to time of detection and the expression of hormone receptor is used as an indicator of adjuvant hormonal treatment to inhibit tumor growth. Thirty percent of breast cancer patients without lymph node metastasis have been shown to have tumor recurrence within 5 years (3). Among these patients, therefore, it is extremely important to screen out those who should receive adjuvant chemotherapy. Recently, there has been a strong tendency to apply adjuvant chemotherapy with smaller breast cancers, and the majority of the breast cancer patients take the adjuvant chemotherapy. In breast cancer patients, there is frequently an early distant metastasis without local recurrence, but with many bone marrow metastases. There have been many studies to unravel micrometastasis in bone marrow, because of the strong tendency for bone marrow invasion in these patients (4–7). To detect micrometastasis, immunohistochemistry and RT-PCR are usually used, however, the former cannot analyze quantitatively and it is very difficult to detect a tumor when it is very small (8). As a marker of bone marrow metastasis in this study, we used keratin-19, which is only expressed in epithelial cells and not in normal bone marrow cells. There are keratin-8, -18 and -19, but keratin-8 and -18 show high false-positive rates. Keratin-19 has been widely studied as a marker of micrometastasis, because it has a high specificity for breast cancer cells and reasonable specificity for bone marrow and blood.
In 1981, Dearnaley et al. showed micrometastasis to bone marrow with no clinical evidence of metastasis (9). They reported 10 cases of micrometastasis to bone marrow among 24 cases and patients with micrometastasis to bone marrow were found to have earlier recurrence than other patients. In addition, among prognostic factors, micrometastasis to bone marrow was seen in 23% of the patients with recurrent tumor (10). Also, they proposed micrometastasis to bone marrow as a prognostic factor, because 85% of the patients with micrometastasis to bone marrow showed cancer recurrence after 9.5 years follow-up, in contrast to 31% of the patients without micrometastasis (11,12).
RT-PCR is a method to determine proteins to find a specific protein that is contained in a specific cell. mRNA is first converted into cDNA and then multiplied. Schoenfeld et al. first employed RT-PCR to detect axillary lymph node metastasis of cancer (13) and Kruger et al. reported bone marrow metastasis in 14 cases among 24 breast cancer patients (14). Vannucchi et al. reported that breast cancer cells were found among stem cells which were used in peripheral blood stem cell transplantation (PBSCT) after high-dose chemotherapy and some patients treated with PBSCT showed early cancer recurrence, suggesting that patients with micrometastasis to bone marrow have greater chance of cancer recurrence (15). Recently, however, Braun et al. reported that micrometastasis to bone marrow is an independent prognostic factor after immunohistochemical study of 150 patients (16).
In the last decade, faster and more precise quantitative and real time RT-PCR methods have been developed (17,18). However, the existence of keratin-19 pseudogene leads to false positives: this pseudogene shows an amino acid sequence similar to that of keratin-19 and can be expressed in non-epithelial tissue (19–23). In the present study, in the case of external primer, the designed primer had two different nucleotides (749, 754 nucleotides) in sense primer and three different nucleotides in antisense primer. In the case of internal primer, four different nucleotides (783, 793, 794, 800) in sense primer and two different nucleotides in antisense primer were designed. These primers are expected to make the pseudogene different from the primer so that the pseudogene has no effect (Table 1).
Further, the nested RT-PCR used in this study had high sensitivity owing to the use of repeated PCR. Recent studies on breast cancer micrometastasis using the nested RT-PCR method indicated 42–49% micrometastasis to bone marrow, showing a higher positive rate than in studies carried out by RT-PCR (24,25). Our results showed a 51% positive rate, i.e. a higher sensitivity than the other methods. However, it is very difficult to explain this difference because our results for the sensitivity test were similar to the others (only one cancer cell could be found among 106 cells).
There were four DCIS cases in the present study and one case among them showed positive expression of keratin-19 mRNA by nested RT-PCR, possibly due to a false positive. Nevertheless, this result might have some significance, since early recurrence of breast cancer has been observed only in the group positive to keratin-19.
In this study, patient treatment and follow-up were carried out in the same manner without any consideration of the RT-PCR results. Comparing various important prognostic factors for micrometastasis to bone marrow, cancer recurrence in stages 2 and 3 was found to be a reasonable prognostic factor. It is well known that lymph node metastasis and expression of hormone receptor and pathological characteristics are important prognostic factors (26). However, in our study there was no statistically significant difference in patient survival. This might have been due to a low recurrence rate and fewer cases. Further studies should be carried out to develop effective guidelines for treatment by reducing the false-positive rate. Several useful technical tips, such as minimization of the chance of contamination from skin and use of mammoglobin, which has specificity for breast cells, could be applied to reduce the false-positive rate. Unfortunately, however, mammoglobin is not expressed in MCF 7 cells (27,28). So far, one positive result has been found in normal bone marrow aspirates. We thought that contamination from skin had induced a false-positive result. In fact, it is very difficult to obtain bone marrow from the normal population, so control samples were limited. This limitation prevents the evaluation of accurate specificity. Further studies should be continued to overcome this problem.
In conclusion, the nested RT-PCR of keratin-19 for micrometastasis of breast cancer showed high sensitivity for micrometastasis of bone marrow. However, one DCIS patient showed a false-positive result, suggesting a problem in specificity and the need for improvement. Every early recurrent breast cancer patients in the present study showed a positive reaction in nested RT-PCR with statistical significance. To be useful as an independent prognostic factor of nested RT-PCR in breast cancer patients, further studies with more cases and longer-term follow-up are required.
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FOOTNOTES |
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+ For reprints and all correspondence: Hee Boong Park, Department of Surgery, Ajou University School of Medicine, Suwon 442-749, Korea. E-mail: parkhb@ajou.ac.kr
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Received January 8, 2003; accepted March 17, 2003
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