Japanese Journal of Clinical Oncology

Introduction

Over 100 years have passed since the standard surgical treatment of breast cancer consisted of removal of the primary tumor and axillary lymph node dissection (ALND) (1). Recently, breast-conserving surgery (BCS) with ALND have been considered indicated for early breast cancer in Japan and also in Western countries (2). ALND results in the clearance of infiltrating cancer cells and provides information on an important prognostic factor, but it is followed by morbidity with axillary pain, numbness and lymphedema in about 10-25% of women (3). In addition, half or more patients have histologically node-negative breast cancer, but lymph node metastasis could not be predicted precisely by preoperative diagnosis (4-7). Recent retrospective analyses have shown that ALND made little influence in decision-making regarding adjuvant therapy in subgroups of early breast cancer, because it was based on the clinicopathological characteristics of the primary tumor (8,9). The management of the axilla in patients with early breast cancer, especially with clinically node-negative breast cancer, is currently a matter of controversy.

The first lymph nodes draining a particular tumor are referred to as the sentinel lymph nodes (SLNs). The histological characteristics of the SLNs are hypothesized to predict the histological findings in the remaining regional lymph nodes. Lymphatic mapping is the first step in identifying the SLNs and the SLN sampling technique is called sentinel node biopsy (SNB). Although SLN detection was first described in penile carcinoma in 1977 (10), SNB has been successfully reported in melanoma and breast cancer since the early 1990s (11-14). A worldwide feasibility study on SNB is currently under way. Many investigators are trying to perform lymphatic mapping and SNB using certain types of blue dye and/or radiolabeled colloid (13-22). At the Division of Breast Surgery, NCCHE, SNB with indigocarmine has been assessed since January 1998. This is the first report on SNB by this method.

Patients and Methods

Eighty-six female patients with stage 0-IIIB breast cancer according to the UICC criteria underwent modified radical mastectomy or BCS with ALND between January and July 1998, including two patients with synchronous bilateral breast cancer. Informed consent with SNB was also obtained before the surgical procedure. Ultimately, a total of 88 cases were evaluated in this series. All cases were diagnosed as malignant by aspiration biopsy cytology, excisional biopsy or intraoperative tumor biopsy.

Under general anesthesia, 4-5 ml of indigocarmine (4 mg/ml) (Daiichi Pharmaceutical, Tokyo, Japan) was injected subcutaneously at two or three sites around the primary tumor and the breast lesions were rubbed well. If the primary tumor had already been excised, the dye was injected near the scar. In patients undergoing total mastectomy, first the whole breast was removed from the pectoralis major muscle. When axillary the fat pad was exposed, the SLNs were identified after lymphatic mapping. Usually blue-staining lymph nodes or dye-filled lymphatic tracts were easily identified in a few minutes. Whenever blue-staining afferent lymphatic tracts were traced to lymph nodes partly stained blue, they were excised as SLNs. After the SLNs were examined, ALND was completed up to Levels I and II or more. In patients undergoing BCS, partial mastectomy was performed 15 min after subcutaneous injection of indigocarmine and then SNB was performed. If the primary tumor was located in the inner or lower region of the breast, ALND was performed through a separate skin incision following SNB.

The pathological diagnosis was based on examination of paraffin-embedded hematoxylin-eosin-stained sections of the primary tumor and all axillary lymph nodes. In some cases, SLNs were immediately diagnosed histologically by frozen-section examination. Histological grade was determined based on the number of mitoses and architectural and cytological atypia by the Bloom and Richardson grading system with modification (23). Estrogen receptor (ER) and progesterone receptor (PR) in the cytosol fraction were determined by enzyme immunoassay (Otsuka Assay Laboratory, Tokushima, Japan). The upper limit cut-off values of the ER and PR assay are 13 and 10 fmol/mg protein, respectively.

Statistical significance was determined by using the chi-squared test or Fisher's exact probability test for differences between the identification of SLNs and the clinicopathological factors of breast cancer.

Results

The age of the patients ranged between 30 and 81 years (median, 53 years). Forty premenopausal and 46 postmenopausal women were included. Twenty-six cases (30%) were stage 0 or I breast cancer, 47 cases (53%) stage IIA or IIB breast cancer and 15 cases (17%) stage IIIA or IIIB breast cancer. Clinical tumor size ranged between 0.0 cm, in T0 breast cancer, and 12.0 cm (median, 3.0 cm). Fifty-five cases (63%) were treated by modified radical mastectomy and 33 cases (37%) by BCS. No postoperative complications, including tattooing in the dye injection area, were recorded in any of the cases, but most patients had green-stained urine a few hours after the dye injection. SLNs were detected in 65 (74%) of the 88 cases. All of the SLNs belonged to Level I in the axilla. The number of SLNs ranged between one and seven nodes (mean, 2.0; median 2.0 nodes), while the number of the remaining axillary lymph nodes ranged between eight and 55 nodes (mean, 21; median, 20 nodes). Table 1 shows the correlations between identification of SLNs and the patients' characteristics. None of the correlations were significant, except with type of surgery. SNB resulted in failure in 16 (48%) of the 33 cases treated by BCS. However, the SNB identification rate increased with surgical experience during the 7-month period. SLNs were detected in 12 (67%) of the 18 cases during the final 3 months, but in only five (33%) of the 15 cases during the first 4 months. The percentage of all cases identified was 72% during the first period and 76% during the last period. No correlations were found between identification of SLNs and the pathological characteristics of the breast cancer either (Table 2).

Table 2. Sentinel node biopsy and pathological features

	No. of cases (%)				P value ([chi]2-test)
	Cases in which SLNs were identified		Cases in which SLNs were not identified
Nodal metastases
0	36	(73)	13	(27)	0.964
1-3	14	(74)	5	(26)
4-9	7	(70)	3	(30)
[ge]10	8	(80)	2	(20)
Histological grade
I	14	(64)	8	(36)	0.114
II	30	(86)	5	(14)
III	21	(68)	10	(32)
Histological subtype
NIDC	3	(75)	1	(25)	0.200
IDCPIC	8	(100)	0	(0)
IDC	44	(69)	20	(31)
ILC	4	(67)	2	(33)
Others	6	(100)	0	(0)
Lymphatic invasion
Ly-	37	(69)	17	(31)	0.234
Ly+	28	(82)	6	(18)
Vascular invasion
V-	35	(69)	16	(31)	0.286
V+	30	(81)	7	(19)
Estrogen receptor
ER+	21	(78)	6	(22)	0.845
ER-	37	(73)	14	(27)
Unknown	7	(100)	3	(30)
Progesterone receptor
PR+	24	(73)	9	(27)	0.920
PR-	34	(76)	11	(24)
Unknown	7	(70)	3	(30)

NIDC, non-invasive ductal carcinoma; IDCPIC, invasive ductal carcinoma with a predominantly intraductal component; ILC, invasive lobular carcinoma.

Table 3. Histological examination of sentinel lymph nodes (sn) in the axillary lymph nodes (ax)

	No. of cases (%)
	Negative ax		Positive ax
Negative sn	36	(100)	4	(14)
Positive sn	0	(0)	25	(86)
Subtotal	36	(100)	29	(100)

Table 4. Sentinel lymph node (sn) positivity in 29 cases of node-positive breast cancer

	No. of cases with positive axillary lymph nodes
	1	2	3-9	[ge]10
Negative sn	3	0	1	0
One positive sn	8	2	5	6
Two positive sn	0	1	1	1
Three positive sn	0	0	0	1

Table 5. Sentinel node biopsy in clinically node-negative and histologically node-positive breast cancer

No. of cases	14
No. of sentinel lymph nodes excised	26
No. of positive sentinel lymph nodes	10
Positive rate	38%
No. of non-sentinel lymph nodes excised	298
No. of positive non-sentinel lymph nodes	23
Positive rate	8%

Histological examination revealed negative SLNs in 40 cases, including four cases with non-sentinel-node-positive breast cancer (Table 3). These false-negative cases consisted of three cases with one positive non-sentinel lymph node in Level I and one case with five positive non-sentinel lymph nodes in Level I (Table 4). In nine (31%) of the 29 cases with node-positive breast cancer, SLNs were the only lymph nodes affected and the non-sentinel lymph nodes were all negative. In 14 cases with clinically node-negative and histologically node-positive breast cancer, the positivity of the involved lymph nodes was significantly higher in SLNs than in the non-sentinel lymph nodes (p < 0.001, chi-squared test) (Table 5). Finally, axillary lymph node status was accurately predicted in 61 (94%) of the 65 cases and SLNs specificity and sensitivity were 100 and 86%, respectively.

In 35 cases, SLNs were histologically diagnosed by immediate frozen-section examination and the SLNs were negative in 27 and positive in eight cases. Among the 27 cases, the final histological examination resulted in 24 cases with negative SLNs, but three cases with microfoci of metastatic cells in the permanent sections of SLNs. There was concordance in 32 (91%) of the 35 cases.

Table 6. Review of sentinel node biopsy in breast cancer

First author	Year reported	Technique	No. of cases with SLNs identified		No. of cases with SLNs alone affected		Sensitivity (%)	Accuracy (%)
Krag13	1993	RC	18/22	(82%)	3/7	(43%)	100	100
Giuliano14	1994	Dye	114/174	(66%)	16/42	(38%)	88	96
Albertini15	1996	RC + dye	57/62	(92%)	12/18	(67%)	100	100
Veronesi16	1997	RC	160/163	(98%)	32/85	(38%)	95	98
Giuliano17	1997	Dye	100/107	(93%)	28/42	(67%)	100	100
Pijpers18	1997	RC	34/37	(92%)	7/11	(64%)	100	100
Galimberti19	1998	RC	238/241	(99%)	39/109	(36%)	95	98
Cox20	1998	RC + dye	440/466	(94%)	NA		99	100
O'Hea21	1998	RC + dye	55/59	(93%)	NA		87	95
Borgstein22	1998	RC	122/130	(94%)	26/44	(59%)	98	99
Present study	1998	Dye	65/88	(74%)	9/29	(31%)	86	94

RC, radiolabeled colloid; NA, not available

Discussion

Intraoperative lymphatic mapping and SNB were performed by using indigocarmine, a dye used for a classical urination test involving ureterocystoscopy. The SNB results reported in the past are shown in detail in Table 6. Many investigators have described excellent results of lymphatic mapping and SNB. Their SLN identification rates exceeded 90% (15-22), compared with 74% in our study. In Western countries, isosulfan blue vital dye (Lymphazurin) is usually used for lymphatic mapping (15,17,20,21), but it is not available in Japan. Although Lymphazurin and indigocarmine are aqueous, Lymphazurin permeates the lymphatic vessels in breast parenchyma more easily than indigocarmine. Nevertheless, this initial experience was impressive, because the sensitivity and accuracy in the present study with blue dye were similar to those reported in other first series (14,21). In some studies, high identification rates were obtained by a combined method using blue dye and radiolabeled colloid (15,20,21). Giuliano and co-workers (14,17) demonstrated that surgical procedures involve a learning curve. This learning curve was clearly shown in the present cases in which BCS was performed.

There were four false-negative cases, because they were SLN-negative and non-sentinel-node-positive breast cancers. Sensitivities reported previously have ranged from 87 to 100% (Table 6). There are several reasons for the false-negative results. First, the surgical technique of SNB needs to be improved and standardized. Surgical experience reduced the incidence of false-negative cases (14). Second, some false-negative cases were characterized by having multifocal tumors or prior excision of the primary tumor (16,19-21). Multifocal tumors were seen in one of the false-negative cases in our series. Such cases may be incapable of intraoperative lymphatic mapping, because of unexpected drainage by one or more lymphatic channels or impaired lymphatic flow. Third, many investigators have reported skip metastases to the upper levels in the axilla (24). The frequency of positive Level II or positive Level III with negative Level I has ranged from 1.5 to 29% or from 0.2 to 10%. According to the SLN hypothesis of breast cancer, however, skip metastases to upper levels may be caused by an unusual lymphatic channel. All of the SLNs in this series belonged to Level I, while the SLNs in Level II but not Level I were identified in other studies (14,15). Fourth, there were micrometastases in SLNs and non-sentinel lymph nodes (14,19,25). Routine histological examination of SLNs has limitations and some investigators emphasize immunohistochemical examination to detect micrometastases (16,20,25). The reliability of intraoperative frozen-section examination of SLNs was found to be another problem (16,19). False-negative rates, including the present study, have ranged from 11 to 24%. Histological, immunohistochemical and molecular biological examination of SLNs is being assessed worldwide and also being discussed between our Division and the Pathology Division at NCCHE.

There are many issues regarding lymphatic mapping and SNB. As mentioned above, there are many different ways to identify SLNs with blue dye, radiolabeled colloid (sulfur colloid) or both (13-22), for example, differences in the site of injection of the dye or colloid, the dose of radioactivity used, the interval between dye injection or lymphoscintiscan and SNB and various surgical procedures for SNB have been used. In addition, exposure to radiolabeled colloid must be safe for surgeons, pathologists and other paramedical personnel. SNB with radiolabeled colloid is still experimental in Japan, because safety criteria have yet to be decided. The theoretical radiation exposure to low-dose technetium-99m-labeled colloid is extremely low, compared with annual natural irradiation (26). In any event, lymphatic mapping and SNB with radiolabeled colloid must be approved by the institutional review board at each hospital. SNB is a promising surgical technique in patients with clinically node-negative breast cancer. SLNs are representative of the remaining axillary lymph nodes. The SLNs in some cases were the only lymph nodes affected (Tables 4 and 6) and lymph node positivity was significantly higher in the SLNs than in the non-sentinel lymph nodes in clinically node-negative and histologically node-positive breast cancer (Table 5). Hence SNB may be a reasonable alternative to unnecessary ALND for local control of early breast cancer.

In conclusion, SNB with indigocarmine was found to be feasible. In the near future, SNB is expected to be performed more successfully by using a combination of blue dye and radiolabeled colloid. Further study is required regarding the guidelines for lymphatic mapping and SNB. Finally, outcome will be evaluated in a randomized clinical trial in early breast cancer patients, comparing SNB with ALND.