Departments of 1 Pathology, 2 Surgical Oncology and 3 Medical Oncology, University General Hospital of Heraklion, Crete, Greece
* Correspondence to: Dr E. N. Stathopoulos, Department of Pathology, Medical School, University of Crete, PO Box 2208, 71003 Heraklion, Crete, Greece. Tel: +30-2810-394692; Fax: +30-2810-394694; Email: stath{at}med.uoc.gr
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Abstract |
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Patients and methods: SLNs and ALNs from 111 patients with operable stage III breast adenocarcinoma were evaluated for the presence of tumor cells by hematoxylineosin (H&E) staining and, if negative, by immunohistochemistry (IHC) using an anti-CK-19 antibody. Peripheral blood was also analyzed for the presence of CK-19 mRNA+ cells by nested RTPCR, before the initiation of adjuvant treatment and in CK-19 mRNA+ patients following the completion of adjuvant chemotherapy and hormonal treatment.
Results: After both H&E staining and IHC analysis, 29 (26%) patients were ALN negative (N0). In 78 (70%) patients H&E staining and in four (3.6%) IHC analysis revealed tumors cells, and these patients were considered as ALN positive (N+). Peripheral blood CK-19 mRNA+ cells were detected in nine (31%) out of 29 N0 and in 31 (38%) out of 82 N + patients (P=0.5) before any adjuvant treatment. Adjuvant chemotherapy and hormone treatment resulted in the disappearance of the CK-19 mRNA+ cells in all N0 patients and in 15 out of 31 N + patients. After a median follow-up of 40 months, all the N0 CK-19 mRNA+ patients were relapse-free whereas four (13%) N + CK-19 mRNA+ patients had relapsed.
Conclusions: Direct hematogenous dissemination of occult tumor cells may occur in a substantial proportion of patients with early-stage breast cancer. The prognostic implication of the detection of these cells requires long follow-up periods and further studies.
Key words: breast cancer, CK-19 mRNA+ cells, hematogenous spread, micrometastatic disease, occult tumor cells, sentinel lymph nodes
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Introduction |
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Several studies have demonstrated that epithelial cells, derived from the initial tumor, can be identified in bone marrow aspirates or/and peripheral blood of patients with breast cancer [1019
]. The malignant origin of these cells was determined by the presence of chromosomal abnormalities, the overexpression of c-erbB2/neu oncogene and their in vitro growth [13
, 14
, 20
]. These occult tumor cells could be identified either immunohistochemically using antibodies against various specific proteins or by RTPCR assay for the detection of mRNA coding for specific epithelial markers. Large prospective studies have shown that the detection of occult malignant cells represents an independent poor predictive and prognostic factor for disease-free interval and overall survival [12
, 14
17
, 19
, 21
].
The metastatic cascade is known to involve multiple steps. Tumor cells must first penetrate into the vasculature of the primary tumor, survive in the circulation, migrate at other sites, grow in the target organ and, finally, induce angiogenesis [14]. Although this is a rather simplistic and general model of tumor cell dissemination, it seems that in breast cancer the main route of metastatic spread is the lymphatic vasculature, with neoplastic cells first migrating in the regional lymph nodes and then spreading to the blood circulation [5
]. However, the incidence of direct hematogenous spread of cancer cells that bypasses the lymphatic vasculature seems to be underestimated in daily clinical practice.
Lymphatic mapping and sentinel lymph node (SLN) biopsy has been introduced as a surgical operation of minimal morbidity that can accurately identify axillary lymph node (ALN)-negative patients and thus spare them the morbidity of axillary lymph nodes dissection [2224
]. On the other hand, SLN biopsy offers the possibility of a more detailed examination leading to the identification of ALN-positive patients who otherwise would be considered as negative by routine pathologic examination. The SLN is defined as the first node to receive drainage from the tumor; its evaluation could provide a unique opportunity to study the natural history of the disease. Indeed, the evaluation of the status of SLN and ALNs in relation to the presence or absence of occult malignant cells in the peripheral blood or the bone marrow of early-stage breast cancer patients may determine the route of spread of tumor cells in these particular patients.
In order to investigate the incidence of direct hematogenous spread of cancer cells in patients with early stage breast cancer, we studied the presence of occult tumor cytokeratin-19 (CK-19) mRNA+ cells in the peripheral blood in relation to the status of SLNs and ALNs. Our hypothesis was that if SLNs and ALNs were negative, then the cancer cells detected in the peripheral blood should have escaped directly into the hematogenous vasculature.
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Patients and methods |
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Detection of SLN
The technical details of SLN identification have been previously reported [25]. In brief, Isosulfan Blue (Patent Blue Violet, Sigma Chemical, St Louis, MO, USA) was used either alone (n=81) or in combination with nanocolloid (Nanocoll; Amersham, Cygne, Eidhoven, The Netherlands) (n=30) using intraoperatively hand-held gamma-detector (Navigator GPS; USSC, USA). The SLN was identified the first lymph node to receive the stain. In the case of more than one node with separate afferent lymph nodes, all were marked as SLN. In the case of a block of stained lymph nodes, an effort was made to identify the first draining lymph node among them. If the effort was unsuccessful, the whole block was marked as SLN. When the probe was used, SLN was identified as the lymph node with an in vivo radioactive count of at least three times that of the background count. If, after the removal of the SLN, the counts of the operative bed were >150% that of the background, the dissection was continued in search of additional SLNs. In all but three patients the SLN biopsy was followed by a level III axillary clearance through the same incision.
Sectioning of lymph nodes and histological examination
SLNs were embedded in toto. Those of more than 3 mm in greatest dimension were longitudinally bisectioned through the hilum, and further cut in parallel to the original section so that the thickness of each yielded slice was 23 mm. Smaller lymph nodes (up to three) were submitted without previous gross sectioning. Tissue slices were fixed in buffered formalin 10% for up to 24 h, routinely processed and paraffin-embedded. Microscopic tissue sections of 3 µm thickness (on negatively charged SuperFrost Plus glass slides) were obtained from three levels of each paraffin block, separated by 30 µm, and examined after hematoxylineosin (H&E) staining and immunostaining as stated below. H&E and immunostaining were performed on adjacent tissue sections. All ALNs identified by palpation of the specimen of the axilla were collected, fixed and further processed as for the SLNs. Micrometastases were considered to be metastatic foci measuring 0.22.0 mm, in greatest diameter, in immunohistochemically stained tissue sections. Smaller foci were characterized as isolated cells (minute micrometastases, micro-micrometastases). Extranodal tissue or vessel infiltrations were not considered as nodal metastases. Metastatic foci in the nodal capsule were accepted as nodal metastases. All histological specimens were evaluated and scored independently by two pathologists (E.N.S. and M.K.)
Immunostaining for CK-19
A monoclonal mouse antihuman CK-19 antibody (DAKO-CK19, Code#M888; DAKO A/S, Glostrup, Denmark) was used at a dilution 1/50 for 60 min, after unmasking by heating for 20 min, in citrate buffer, pH 6, in a microwave oven at 500 W. The immunostaining method applied was a polymer method based on the DAKO EnVision System, Alkaline Phosphatase, Universal (Code#K1396) with Fast Red substrate-chromogen, according to the manufacturer's instructions. Counterstaining was done with hematoxylin (Harris), and coverslipping with an aqueous-based mounting medium (Glycerol, cook # C-0563, Carpinteria, CA). Known positive and negative controls were always used.
Detection of CK-19 mRNA+ cells in peripheral blood
The method of blood collection, processing and performance of the nested RTPCR reaction has been previously described in detail [19]. In short, blood samples (10 ml in EDTA) were obtained at the middle of vein puncture to avoid contamination with epidermal epithelial cells and after dilution in phosphate buffered saline (PBS) and centrifugation through Ficoll-Hypaque, total RNA isolation was performed. The isolated RNA was dissolved in diethylpyrocarbonate-treated water and stored at 80°C until used. RNA integrity was tested by PCR amplification of the ß-actin housekeeping gene. RNA prepared from the MCF-7 and ARH-77 human tumor cell lines, was used as positive and negative control, respectively.
Reverse transcription of RNA was carried out with the Thermoscript RTPCR system (Gibco). Two different PCR reactions with the respective negative controls were performed with each sample in order to amplify fragments of CK-19 and ß-actin. The sequences of primers used (Genset, Paris, France) have been previously reported [19]. These primers extend across at least an intron, so an eventual DNA contamination would not pose a significant problem. The corresponding sizes of PCR products were 745 and 145 bp for CK-19 and ß-actin, respectively.
Statistical methods
Continuous variables are expressed as the median value with the range given in parentheses. Comparisons between proportions were made using the Pearson's 2 statistical test.
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Results |
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HC staining of SLNs and ALNs
All H&E-negative SLNs and ALNs were immunostained using an anti-CK-19 monoclonal antibody (mAb). Four (12%) out of 33 patients with H&E-negative SLNs were stained positively with the anti-CK-19 mAb and these patients were upgraded in stage. Therefore, based on the H&E and IHC staining, 82 (74%) patients were considered to have N+ (stage II) and 29 (26%) N0 (stage I) disease. Moreover, four (7%) out of 61 patients with H&E-negative ALNs stained positive with anti-CK-19 mAb; two of these patients had at least one H&E-positive SLN whereas the other two patients had SLN stained positive with the anti-CK-19 mAb. Four SLNs and 11 ALNs which were H&E-negative were revealed to be positive by IHC.
Detection of peripheral blood CK-19 mRNA positive cells according to the status of SLNs and ALNs
CK-19 mRNA-positive cells in the peripheral blood could be detected in 40 out of 111 (36%) patients before the initiation of any adjuvant chemotherapy or hormone treatment. Peripheral blood CK-19 mRNA+ cells could be detected in 9 (31%) out of 29 N0 and in 31 (38%) out of 82 N + patients (P=0.514) (Table 2). Table 3 compares the incidence of CK-19 mRNA detection in the peripheral blood of patients according to the involvement of SLNs or/and ALNs. Peripheral blood CK-19 mRNA+ cells could be detected in 14 (50%) SLN+/ALN, in 14 (29%) SLN+/ALN+ and in three (50%) SLN/ALN+ patients. There was no statistical difference in the incidence of CK-19 mRNA+ cell detection between SLN+/ALN and SLN+/ALN+ (P=0.069), SLN/ALN+ and SLN+/ALN+ (P=0.300) or between SLN/ALN+ and SLN+/ALN (P=1.000) groups.
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Discussion |
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The SLN is theoretically the first ALN to receive drainage from the tumor and, thus, it should be the first lymph node to be involved by tumor cells. The incidence of CK-19 mRNA+ cell detection in the peripheral blood was 29% in patients with SLNs+/ALNs+ and 50% in those with SLNs+/ALNs, a difference that is not statistically significant. Moreover, the relapse rate was similar in the two groups. Previous studies revealed that the detection of occult tumor cells in the bone marrow or the peripheral blood of N + patients also has a poor prognostic value, as does the number of involved lymph nodes [12, 14
19
]. Moreover, these studies have demonstrated that there is no consistent relationship between the detection of occult tumor cells in the bone marrow or the peripheral blood and other commonly used pathological prognostic factors [14
19
]. In one study there was even a significant association of CK-19 positivity by RTPCR in the blood with distant metastatic versus both node-negative and node-positive disease, but not with any other histopathological parameter examined [28
].
So far, after a median follow-up period of 40 months no distant metastases were observed in our N0 patients, despite the presence of CK-19 mRNA+ cells in the peripheral blood. This may be attributed to the fact that adjuvant chemotherapy could decrease the number of CK-19 mRNA+ cells in the peripheral blood to undetectable levels in seven out of nine patients, whereas subsequent adjuvant hormone treatment could eliminate peripheral blood CK-19 mRNA+ cells in the remaining two patients (Table 4). Alternatively, the lack of relapse may be explained by the relatively short follow-up time for the nine N0/CK-19 mRNA+ patients (median 29 months). In the N + patients, adjuvant chemotherapy and hormone treatment failed to decrease the number of CK-19 mRNA+ cells in the peripheral blood to undetectable levels in 26% of the patients and four of these patients relapsed during adjuvant hormone treatment. Although the duration of follow-up period may be an important factor for the validation of the prognostic and predictive value of the peripheral blood CK-19 mRNA+ cells in such studies, we cannot exclude that this observation may suggest that CK-19 mRNA+ cells in N0 patients are more sensitive to chemotherapy and hormone treatment than those observed in N+ patients; such a hypothesis, which is probably related to phenotypic changes of occult tumor cells [2932
], is in agreement with the tumor dormancy hypothesis [33
, 34
]. In addition, we cannot exclude that the tumor burden of patients with N+ disease is significantly higher than that of patients with N0 disease and, therefore, more likely to contain subsets of tumor cells that are resistant to chemotherapy and/or hormone treatment. Therefore, the presented data seem to indicate that there are at least two different subpopulations of occult tumor cells: the first one corresponds to cells that are sensitive to adjuvant chemotherapy and/or hormone treatment and the second one to cells that are resistant to adjuvant treatment. Whether their sensitivity to adjuvant chemotherapy and/or hormone treatment is related to their hematogenous or lymphatic circulation remains an open question. Nevertheless, according to this hypothesis the chemotherapy and hormone treatment resistant tumor cells may switch to an angiogenic phenotype [35
, 36
] through multiple regulatory factors produced by tumor stromal cells and/or infiltrating blood cells [37
40
], thus escaping dormancy and leading to clinically evident metastatic disease.
In conclusion, the data reported in the present study clearly indicate that the hematogenous spread of tumor cells in patients with breast cancer may be an early phenomenon occurring before the involvement of the regional ALNs. Adjuvant chemotherapy and hormone treatment might eliminate or decrease the number of occult tumor cells to undetectable levels. Since there is no cure for patients with clinically overt metastases, the only possibility to cure these patients is to try to eliminate occult tumor cells early during the metastatic process, by combining different therapeutic approaches such as chemotherapy, hormones and targeted biological treatments. For this purpose it is important to further understand the mechanisms of tumor cell dissemination and to define specific groups of breast cancer patients who have an increased risk of relapse.
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Acknowledgements |
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Received for publication May 25, 2004. Revision received September 1, 2004. Accepted for publication September 10, 2004.
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