10.1245/ASO.2005.07.013
Annals of Surgical Oncology 12:497-503 (2005)
© 2005 Society of Surgical Oncology
Correlation Between Concordance of Tracers, Order of Harvest, and Presence of Metastases in Sentinel Lymph Nodes With Breast Cancer
Noriaki Wada, MD1,
Shigeru Imoto, MD1,
Chisako Yamauchi, MD1,
Takahiro Hasebe, MD2,
Atsushi Ochiai, MD2 and
Satoshi Ebihara, MD3
1 Breast Surgery Division, National Cancer Center Hospital East, 6-5-1, Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan
2 Pathology Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan
3 Head & Neck Surgery Division, National Cancer Center Hospital East, 6-5-1, Kashiwanoha, Kashiwa, Chiba, 277-8577, Japan
Correspondence: Address correspondence and reprint requests to: Noriaki Wada, MD; E-mail: nowada{at}east.ncc.go.jp.
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ABSTRACT
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Background: There are various methods for the detection of sentinel lymph nodes (SLNs) in breast cancer by using a combined method with blue dye and radioisotope (RI) tracers. The purpose of the study was to reveal any correlation between concordance of the tracers and the order of harvest with the presence of metastases in SLNs.
Methods: The outcomes were reviewed in 408 cases with stage 0 to II breast cancer; the combined method was used in which blue dye and RI were injected subcutaneously around the tumor. The radioactivity and blue staining in each harvested SLN were checked.
Results: In 330 cases (81%), SLNs contained both blue dye and RI tracers (blue-hot cases), and in 42 (10%) and 31 (8%) cases, the SLNs contained only the blue stain (blue-only cases) and only RI (hot-only cases), respectively. The overall metastatic rate was 25% on a patient basis. Blue-only cases had a higher rate (42%) of metastasis than hot-only cases (14%). The rate of nodes containing both blue dye and RI gradually decreased from the first SLNs harvested to the third SLNs harvested. The rate of nodes containing RI only increased with the number harvested, and there was not so much change in the rate of nodes containing blue only.
Conclusions: These data suggest that RI tracer could detect a wide range of SLNs and that the blue dye tracer could efficiently detect SLNs with metastasis. The combined methods compensates for the deficiencies of each method and thus will probably help to prevent missing SLNs.
Key Words: Breast cancer • Sentinel lymph node biopsy • Sentinel node mappings • Blue dye • Radioisotope • Lymph node metastasis
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INTRODUCTION
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Sentinel lymph node (SLN) biopsy (SNB) in breast cancer is a highly sensitive technique for detecting lymph node metastasis in a minimally invasive manner. Because SLNs are the most likely sites of lymphogenic metastasis, the primary benefit offered by SNB is the elimination of axillary lymph node dissection in lymph node–negative patients.
The technique of detecting SLNs commonly uses tracers such as blue dye, radioisotope (RI), or both. Krag et al.1 and Giuliano et al.2 first reported SLN mapping for breast cancer with RI alone and blue dye alone, respectively. Recently, the combined method using both tracers has been reported to give a high identification rate of SLNs with a low false-negative rate. However, there is no uniformity of technique for applying the combined method. In the recent literature, summarized in Table 1
,3–13 investigators have reported identification rates with various manners of injection. There are two main injection sites: areolar or around the tumor with deep intra-parenchymal, subdermal, or intradermal administration. The optimal site and depth for the injection of the combined blue dye and RI tracers are not clear.
With the combined method, lymph nodes detected either by staining blue or containing RI are usually considered SLNs. Most of the harvested SLNs contain both tracers, and there are usually one to three harvested SLNs. However, we sometimes encounter SLNs containing only one of the two tracers. In addition, the correlations among the concordances of both tracers, the order of SLNs harvested, and the presence of metastasis are not still clear. These factors must be considered when SLNs are detected. The purpose of this study was to assess the correlation among these factors by using subdermal injection of blue dye and RI around the tumor.
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PATIENTS AND METHODS
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Between January 1999 and December 2002, the outcomes were reviewed of 442 consecutive patients with stage 0 to II breast cancer who had undergone SNB performed with a combination of blue dye and radio guided methods. No patient had received neo-adjuvant chemotherapy. Patients were excluded from the study if 
4 SLNs were harvested in the axilla (15 cases), if the internal mammary SLN could be removed (8 cases), or if non-SLNs were involved without metastasis in SLNs (5 cases), because these were a small number of cases. A further six patients in whom SLNs could not be identified were also excluded. Finally, a total of 408 cases were analyzed in this study.
Lymphatic mapping and the SNB procedure were explained to the patient, and informed consent was obtained before the surgical procedure. These SNB techniques have been described in detail elsewhere.14,15 Briefly, 30 to 50 MBq (.8–1.4 mCi) of 99mTc human serum albumin and 99mTc tin colloid (Nihon Medi-Physics, Tokyo, Japan) were injected subcutaneously 1 day before the surgical procedure at one to three sites around the primary tumor, but not into the tumor or the biopsy cavity. These radioactive tracers were not filtered. Preoperative lymphoscintigraphy was performed with a large-field scintillation camera.
With patients under general anesthesia, 4 to 5 mL of indigocarmine (4 mg/mL) (Daiichi Pharmaceutical, Tokyo, Japan) was injected subcutaneously at one to three sites around the primary tumor, and the breast lesions were gently massaged. SNB was then performed by using a combination of the presence of blue dye staining and radioactivity detected with a handheld gamma ray detection probe (Navigator; USSC, Norwalk, CT). In our procedure, a skin incision was made axially, and at first, careful dissection was performed for several minutes to search for blue lymphatics draining to a blue-stained lymph node (tract guided). If this was not successful, then the gamma probe was immediately used to find SLNs (probe guided).
All SLNs were submitted for immediate frozen-section diagnosis. While we awaited the results, we performed a partial or total mastectomy. Patients diagnosed as having positive SLNs on the basis of the frozen-section diagnosis immediately underwent an axillary lymph node dissection of level I, II, or higher. However, 42 patients had a backup axillary dissection scheduled in advance as part of a feasibility study, and 10 axillary dissections were performed in keeping with the patients’ wishes, regardless of the result of the frozen-section diagnosis. The SLNs and other dissected non-SLNs were later diagnosed by using permanent sections and routine hematoxylin and eosin staining at a single section.
All harvested SLNs were individually and separately checked for the ex vivo radioactive count, which was recorded, and the degree of blue staining. Successful blue dye localization was defined as a lymph node with visible blue staining, directly contiguous blue-stained afferent lymphatics, or both. Any radioactive node was defined as SLN. We did not adopt a certain threshold over the background.
The categories of SLNs harvested are listed in Table 2. All SLNs were classified into three types of nodes, as follows: those containing both blue dye and radioactivity (blue-hot node), those containing radioactivity alone (hot node), and those containing blue dye alone (blue node).
All cases were divided into four combinations per case according to the types of SLNs. The blue-hot case was defined as including only blue-hot nodes or at least one blue-hot node and other types of nodes. The hot-only case and blue-only case had hot nodes alone and blue nodes alone, respectively. The blue-only/hot-only case was defined as having a mixture of both blue nodes and hot nodes.
We recorded the order and the total number of SLNs harvested. The order of harvest was simply determined by the order in which the SLN was discovered. The first, second, and third SLNs harvested were expressed as sn1, sn2, and sn3, respectively. Moreover, in this study we defined a case with only one SLN harvested as a 1-SLN case and, in the same way, a case with two and three SLNs as a 2-SLNs case and 3-SLNs case, respectively. For example, a 2-SLNs blue-hot case was a patient in whom two blue-hot nodes or one blue-hot and another type of node were identified.
Statistical significance was determined by using a 
2 test with StatMate III (ATMS Co. Ltd., Tokyo, Japan). Differences between groups were considered significant at P < .05.
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RESULTS
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Patient characteristics are listed in Table 3. The age of the patients ranged from 25 to 84 years (median, 54 years). A total of 403 patients underwent SNB for 408 axillary nodal basins. Five patients had bilateral breast cancer, and 35 patients had a prior excisional biopsy. A total of 657 SLNs were removed, with an average of 1.6 lymph nodes per case.
There were 330 blue-hot cases (81%). Hot-only cases and blue-only cases accounted for 42 (10%) and 31 (8%) cases, respectively. There were five (1%) blue-only/hot-only cases. Table 4 shows the metastatic rates associated with categories of SLNs on a patient basis. The overall metastatic rate was 25%. The metastatic rates of blue-hot cases, hot-only cases, and blue-only cases were 25% (83 of 330), 14% (6 of 42), and 42% (13 of 31), respectively. The metastatic rate of blue-only cases was the highest (p = .03). Of 13 metastatic cases in blue-only cases, 6 (46%) had 3 involved lymph nodes in the axilla. Coversely, all six metastatic cases in hot-only cases had only one or two lymph node metastases in the axilla. Two hundred forty-seven (75%) of all 330 blue-hot cases consisted of only blue-hot nodes. Seventy-eight (94%) in 83 involved cases of all blue-hot cases had metastases in blue-hot nodes. Five cases had metastasis in other than blue-hot nodes.
Of the total of 408 cases, 52% (n = 211) accounted for 1-SLN cases; 36% (n = 145) and 13% (n = 52) accounted for 2-SLNs and 3-SLNs cases, respectively. Similar metastatic rates were seen among these three groups (Table 4
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Figure 1 shows the classification of all cases by the relationship between the identified patterns and the total number of harvested SLNs on a patient basis. The rates of blue-hot cases in the 1-, 2-, and 3-SLNs cases were 77% (n = 162), 84% (n = 122), and 88% (n = 46), respectively. On the whole, there was a significant difference in the percentage of identified SLN patterns among groups (P < .01).
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FIG. 1. The relationship between the combination and the number of SLNs harvested on a patient basis. SLN, sentinel lymph node.
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Each lymph node was classified according to the relationship between the type and order of SLNs, as shown in Fig. 2. Of all 657 lymph nodes, the rates of blue-hot nodes, hot nodes, and blue nodes were 67% (n = 442), 20% (n = 134), and 12% (n = 81), respectively. The blue-hot node rate gradually decreased from sn1 to sn3 (sn1, 78%; sn2, 53%; and sc3, 38%). Conversely, there was a trend toward a higher rate of hot nodes for sn3 (48%; n = 25) compared with sn1 (12%; n = 50) and sn2 (30%; n = 59). The rate of change in blue node cases was small. A statistically significant difference in the rate of change was observed among the groups (P < .001).
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FIG. 2. The relationship between the types and the order of SLNs harvested on a lymph node basis. SLN, sentinel lymph node; sn1, the first SLN harvested; sn2, the second SLN harvested; sn3, the third SLN harvested.
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Table 5 shows metastatic rates associated with the order of SLNs harvested on a lymph node basis. In each category, sn1 was most often involved in metastasis of sn1 to sn3. The 1-SLN cases had the highest rate (25%) of metastasis among the three groups, with statistical significance (P = .07). Of a total of 102 cases with metastasis, 90 cases (90 of 102; 88%) were in sn1. Of the remaining 12 cases with metastasis, 10 cases (10 of 102; 10%) were in sn2; and 2 cases (2 of 102; 2%) were in sn3.
A detailed analysis in Fig. 3 shows the percentage of metastatic lymph nodes with the identified pattern of SLNs by the number and order on a lymph node basis. In sn1 of 1-SLN cases, the rates of metastasis in blue node group and the hot node group were 45% and 11%, respectively. There was a significant difference between them (P = .02). Conversely, there was 33% metastasis in the hot node group of 3-SLNs cases, but there was no metastasis in the blue node group of 3-SLNs cases.
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FIG. 3. The percentage of metastatic lymph nodes with the types of SLN by the number and the order of harvest on a lymph node basis. SLN, sentinel lymph node; sn1, the first SLN harvested; sn2, the second SLN harvested; sn3, the third SLN harvested; NS, not significant.
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DISCUSSION
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In this study, we examined the correlation between the concordance of two tracers (blue dye and RI) and the presence of metastasis, considering the order and number of SLNs harvested. This study is the first to evaluate the discordance between tracers and the rate of metastasis and to distinguish between the order and number of SLNs harvested.
Patients with harvested internal mammary lymph nodes, four or more SLNs detected, and false-negative SLNs were excluded because these were rare cases and the analysis became more complicated. Although the false-negative rate is important, we cannot exactly calculate a false-negative rate because this study was not validated by a completion lymphadenectomy if the SNB was negative. The total number of patients who had backup axially lymph node dissection for a feasibility study or because of patients wishes was 52. There was no false-negative case in those patients. All five false-negative cases that were excluded from this study had non-SLN metastases that were accidentally found in level I or in a gland of the breast. When lymph nodes that had neither radioactivity nor blue dye were harvested for suspected metastasis (swelling or hard palpable area), the lymph nodes were not accepted as SLNs in this study. Our previous pilot study, in the same manner, showed that the false-negative rate was 9.5% (2 of 21).15 Furthermore, in the case of the internal mammary SLN, excision of the lymph node was the final procedure just after the axillary lymph nodes were harvested, and the incidence of ipsilateral internal mammary metastasis in the absence of axillary metastasis was low.16
All cases had subcutaneous injection of both tracers overlaying the tumor or the biopsy scar. Apart from the eligible 408 cases, in 6 cases no SLNs could be identified. The identification rate was 99% (408 of 412), for reference. The rates of blue-hot cases on a patient basis and blue-hot nodes of all nodes were 81% and 67%, respectively. The concordance rate was lower than could have been expected. Table 1 summarizes the relationship between the injection sites of dual tracers and the concordance rate in the recent literature. Many investigators were concerned with the concordance rate for a dual-tracer approach, different depth of injection, or separate-site approach. They performed various experiments in pursuit of the highest identification rate. In aggregate, these studies demonstrate that the identification rate with an injection site that includes the subareolar or periareolar approach is a little higher than that with injection around the tumor alone (averaging 98% vs. 95%) and that the concordance rate is considerably high (averaging 92% vs. 75%) (Table 1).
Why the blue dye and RI tracers are not taken up equally into SLNs after injection into nearly the same site around the tumor is not clear. Kern10 mentioned several differences between the dye and radiocolloid in explanations of the discordance, as follows: (1) the molecular weight and the chemical structure,17 (2) the intensity of binding of some kinds of protein to the tracers, (3) the injection time of tracers, and (4) the sensitivity of methods (a handheld gamma probe vs the naked eye). Furthermore, it is difficult to understand how injection into the same site around the tumor could lead to separate drainage pathways to different SLNs.
In this study, discordance between the hot and blue tracer methods occurred in 78 cases. There were more hot-only cases (n = 42; 10%) than blue-only cases (n = 31; 8%). On a lymph node basis, discordance occurred in 215 nodes. The total number of hot nodes (n = 134; 20%) was more than that of blue nodes (n = 81; 12%), and 48% of sn3 were hot-node only. This observation may reflect drainage to secondary lymph nodes. One of the most important reasons for this might be that RI tracer is injected earlier in our procedure: generally 24 hours before the blue dye is injected. A population of small-diameter particles in the unfiltered tin colloid or human serum albumin may easily to migrate to secondary or higher SLNs because of the operation.
Also, the metastatic rate in hot-only cases (14%) was lower than that in blue-only cases (42%). Likewise, on a lymph node basis, there was a considerable difference in metastatic rates between the hot node and blue node groups, particularly in sn1. Thirteen (14%) of 90 positive sn1 lymph nodes were detected by blue dye alone and might have remained undetected if only a gamma probe had been used (Table 5).
There is an important relationship between the number and order of harvest and metastasis, although the order might have been influenced by our manner of searching for SLNs: tract guided (blue dye) and probe guided (RI). A detailed analysis of the metastasis detection rate of RI versus blue dye suggested that blue dye might be more sensitive. The sn1 is especially meaningful. It might be the most suggestive primary lymph node draining from the tumor. In the sn1 of 1-SLNs cases (Fig. 3), the rate of metastasis (45%) in blue node group was extremely high, but there was only 11% metastasis in the hot node group. However, there was 33% metastasis in the hot node group of 3-SLNs cases, but there was no metastasis in the blue node group of 3-SLNs cases. When we detect an sn1 harvested with discordance (hot node or blue node), the meaning (probability of metastasis) changes according to the number of harvest.
Nieweg and Estourgie18 suggested that a bulky metastatic tumor in the true SLN or in the afferent lymphatic channel might actually divert injected tracers into "neosentinel" nodes that do not contain metastasis. A capacity for taking radiocolloids into SLNs may well require some phagocytic activities to achieve a detectable level of radioactivity.19 The small blue dye crystals passively rely on prevailing fluid dynamics for visualization of the lymph node as blue stained. That may be why RI tracer could be more difficult than blue dye tracer to break into the metastatic lymph node. Our experiments clearly revealed that extensive lymph node metastases in the axilla could be detected only with the blue dye method.
In conclusion, the concordance rate between blue dye and RI by using subcutaneous injection around the tumor was 81%. The identification rate, however, remained high (99%). Blue-only cases had a high rate of metastasis. These data suggest that the RI method was capable of detecting a wide range of SLNs and that blue dye tracer could efficiently detect SLNs with metastasis. The combination of these two methods compensates for the deficiencies of each method used individually and, thus, will probably help to prevent missing SLNs during location of axial SLNs in patients with early breast cancer.
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ACKNOWLEDGMENTS
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Supported in part by a Grant for Scientific Research Expenses for Health Labor and Welfare Programs and the Foundation for the Promotion of Cancer Research and by the 2nd-Term Comprehensive 10-Year Strategy for Cancer Control.
Received for publication July 13, 2004.
Accepted for publication January 19, 2005.
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ABSTRACT
INTRODUCTION
