Chinese Medical Journal 2007;120(20):1762-1765
Contribution of minute axillary lymph nodes to accurate staging for patients with breast cancer

JIANG Jun,  HE Qing-qing,  YANG Xin-hua,  LIANG Yan,  FAN Lin-jun,  ZHANG Yi,  GUO Mei-qin

JIANG Jun (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

HE Qing-qing (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

YANG Xin-hua (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

LIANG Yan (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

FAN Lin-jun (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

ZHANG Yi (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

GUO Mei-qin (Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China)

Correspondence to:JIANG Jun,Breast Disease Centre, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China (Tel: . Fax:. E-mail:jcbd@medmail.com.cn)
Keywords
breast cancer; minute lymph node; pathological staging
Abstract

Background  Axillary lymph node metastasis is a very important metastatic pathway in breast cancer and its accurate detection is important for staging tumour and guiding therapy. However, neither the accuracy of routine detection of lymph node in surgical specimens nor the significance of minute lymph node with metastases in breast cancer is clear. A modified method for conveniently detecting minute lymph node in specimens of axillary dissections in patients with breast cancer was used to analyze their influence on staging breast cancer.      
Methods  Lymph nodes in fresh, unfixed, specimens of axillary dissections from 127 cases of breast cancer were detected routinely. Then the axillary fatty tissues were cut into 1 cm thick pieces, soaked in Carnoy’s solution for 6 to 12 hours, taken out and put on a glass plate. Minute lymph nodes were detected by light of bottom lamp and examined by routine pathology.
Results  Lymph nodes (n= 2483, 19.6±8.0 per case) were found by routine method. A further 879 lymph nodes up to 6 mm (781 < 3 mm, 6.9±5.3 per case, increasing mean to 26.5±9.7) were found from the axillary tissues after soaking in Carnoy’s solution. By detection of minute lymph nodes, the stages of lymph node metastasis in 7 cases were changed from pathological node (pN) stage pN0 to pN1 in 4 cases, from pN1 to pN2 in 2 and from pN2 to pN3 in 1. 
Conclusions
 The accurate staging of axillary lymph node metastasis can be obtained routinely with number of axillary lymph nodes in most cases of breast cancer. To avoid neglecting minute lymph nodes with metastases, small axillary nodes should be searched carefully in the cases of earlier breast cancer with no swollen axillary nodes. Treatment with Carnoy’s solution can expediently detect minute axillary nodes and improve the accurate staging of lymph nodes in breast cancer.

Dissection of axillary lymph nodes is an important procedure in breast cancer surgery. Detecting sufficient axillary nodes in specimens of surgical dissections is the precondition of identifying metastases in axillary nodes. The latest staging and therapy guidelines for breast cancer take the number of axillary nodes with metastases as the most important parameter.1-3 Because of the difficulty of routine identification of small lymph nodes in the excised specimen, whether enough lymph nodes are detected to avoid loss of information is still not clear. It is also unknown in which cases the minute lymph nodes with metastases are routinely undetected. Using lypolysis to increase contrast between fatty tissue and lymph nodes we routinely detected macroscopic minute lymph nodes in specimens of axillary dissections of breast cancer patients. The clinical significance of detection of minute axillary nodes in the specimens of axillary dissections of breast cancer is discussed.

METHODS

Subjects
From December 2004 to January 2006, 127 female patients with breast cancer were surgically treated at Breast Disease Centre, Southwest Hospital, Chongqing, China. The age of the patients ranged from 23 years to 77 years (mean 47.6±10.3 years). Clinical staging of breast cancer included stage 0 (the mass in breast was not palpable in clinical examination) in 4 cases, stage I in 16, stage II in 58 and stage III in 49. No distant metastases were found. Pathological types were infiltrating ductal carcinoma in 79 cases, infiltrating lobular carcinoma in 32, carcinoma simplex in 2, intraductal carcinoma in 5, squamous cell carcinoma in 1, breast epithelial- myoepithelial carcinoma in 2, medullary carcinoma in 2, tubular carcinoma in 2, and mucinous carcinoma in 2. The surgical procedures included modified radical mastectomy in 104 cases, modified radical mastectomy plus thoracoscopic internal mammary nodes chain dissection in 9, radical mastectomy plus internal mammary lymph node biopsy via intercostals space in 1, and wide local lumpectomy in 13. All patients were treated with level I, II or III axillary lymph node dissection.

Methods of searching for axillary dissection lymph nodes
After axillary dissection, the surgeons searched routinely for lymph nodes in the fresh tissues. Then the dissected axillary fat tissues were washed to remove the blood, cut into 1-cm thick sections and soaked in Carnoy’s solution (60 ml ethanol, 30 ml chloroform and 10 ml pure ethanoic acid) for lypolysis for 6-12 hours. The volume of Carnoy’s solution was 2-3 times of fat. Then the tissues were taken out, washed and placed on the glass plate above a lamp. After each piece of tissue was spread, the easily identified, minute lymph nodes appeared milky white in colour on a background of incompletely dissolved, semitransparent flaxen fat (Fig. 1). Turning on the light under the glass plate, lymph nodes larger than 1 mm were seen as spots brighter than the fat tissue. The number and sizes of lymph nodes were recorded. The lymph nodes > 1 cm were cut longitudinally into three pieces and embedded with paraffin separately to decrease false negative results. The lymph nodes found with routine method and the minute lymph nodes found after lypolysis were assessed separately, imbedded with paraffin and cut into sections followed by routine pathological examination.

Statistics methods
Paired t test and Excel 2003 software were used for analysis. P<0.05 is considered statistically significant.

RESULTS

HE stained sections of minute lymph nodes found after soaking in Carnoy’s solution clearly showed cell structures, metastatic breast cancer cells and background tissues of lymph nodes. CK-19, ER and PR immunohistochemical staining results were similar to those of lymph nodes detected by routine methods (Figs. 2, 3 and 4). Soaking in Carnoy’s solution had no influence on histopathological observation and immunohistochemical staining of detected lymph nodes.


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Fig. 1. Incompletely lysed semitransparent flaxen fat and white lymph nodes
Fig. 2. Infiltrating ductal carcinoma of breast CK-19 immunohistochemical staining (S-P staining method ×100)
Fig. 3. Infiltrating ductal carcinoma of breast ER immunohistochemical staining (S-P staining method ×200)
Fig. 4. Infiltrating ductal carcinoma of breast PR immunohistochemical staining (S-P staining method ×200)

In the 127 patients with breast cancer, 2483 axillary lymph nodes were found by routine method (4 to 52 lymph nodes per case; mean 19.6±8.0 lymph nodes per case). Another 879 minute lymph nodes were found from the conventionally detected axillary tissues after using fat clearing method (1 to 31 lymph nodes per case, mean 6.9±5.3 lymph nodes per case). So the result of using the two methods together demonstrated 8 to 69 lymph nodes per case (mean 26.5 ±9.7 lymph nodes per case), and the number of detected axillary lymph nodes was increased significantly compared with that by routine method (P<0.0001). Among the 879 minute lymph nodes found after fat clearance, only 41 nodes (4.7%) were > 4 mm in diameter (Table 1).


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Table 1. The size and status of detected axillary lymph nodes of 127 cases after fat clearance.

The detection rate of axillary lymph nodes
The count of lymph nodes were fewer than 10 in 5 cases detected with routine method; reexamination with fat clearance found only 2/5 with fewer than 10 lymph nodes (Table 2).
 

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Table 2. The number of axillary lymph nodes of breast cancer found with different methods (n)

The comparison of positive detection rates of lymph nodes at different clinical stages of breast cancer is shown in Table 3. The routine method found 77 cases with positive axillary lymph nodes, accounting for 60.6% of the total cases; after fat clearance the number increased to 81 cases (63.8%). In addition, in 2 cases of stage 0 breast cancer and 2 cases of stage I who were examined to have negative lymph nodes by the routine method, metastasis was found in minute lymph nodes after reexamination with fat clearance method.
 

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Table 3. Positive rate of detected axillary lymph nodes of different clinical stages of breast cancer (n)

Twenty-six patients were shown with metastatic lymph nodes after fat clearance. pN staging of 7 cases was changed, from pN0 to pN1 in 4 cases, from pN1 to pN2 in 2, and from pN2 to pN3 in 1 (Table 4).
 

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Table 4. Change of pN and axillary lymph nodes metastasis of 7 cases after fat clearance

DISCUSSION

The follow-up of a large number of cases showed that the number of metastatic axillary lymph nodes is the most important independent prognostic factor and that metastatic axillary lymph nodes indicate possible distant spread.4 Enough lymph nodes for pathological examination is a precondition to determine whether there is lymph node metastasis and the number of metastatic lymph nodes. National Comprehensive Cancer Network5 and American Society of Clinical Oncology6 take the number of metastatic lymph nodes as an important basis for breast cancer staging, which has a direct influence on prognosis and the choice of treatment of patients with breast cancer. To ensure the accuracy of clinical staging, as many lymph nodes as possible must be detected. Which situations cause missed diagnosis of metastatic lymph nodes are clinical problems that deserve attention.

To detect 90% of metastasized axillary lymph nodes, the number of axillary lymph nodes dissected must be at least 10.7 The method of macrography and direct palpation of resected specimens is commonly used to seek out lymph nodes. In this series, surgeons of our group using macrography and direct palpation could find most lymph nodes larger than 4 mm from fresh specimens without formalin fixation, but fewer than 10 lymph nodes were found in the axillary specimen in 5 patients. After using fat clearance only 2 out of the 5 cases were found to have fewer than 10 lymph nodes (one with 8 lymph nodes and one with 9 lymph nodes). The positive detection rate of lymph nodes was significantly increased when the fat clearing method was used.

To raise the detection rate of lymph nodes many researchers have been searching for new methods of detecting axillary lymph nodes. In 1948 the fat clearing method was first used to identify lymph nodes from dissected specimens in 87 breast cancer patients treated with radical mastectomy, with the mean number of lymph nodes detected being 30.4.8 Based on this, the fat clearing method has been improved by some researchers. But the procedure is complicated and needs a lot of reagents so it is not widely used.7,9,10 Koren et al11 used fat clearance in axillary tissues resected from 13 breast cancer patients and found lymph nodes larger than 4 mm could be detected, thus TNM staging was upgraded in 4 patients. Some internal reports show stain of axillary lymph nodes with ink injection is used as guidance for dissecting and detecting lymph nodes.12 But this method does not stain all axillary lymph nodes. Therefore, in clinical practice macrography and direct palpation are still widely applied to search for lymph nodes.

In this series, the mean number of lymph nodes found by the traditional method of macrography and palpation from fresh specimens was 19.6 per case, and after fat clearance, the mean number of axillary nodes was increased to 26.5 per case. After tissues are soaked in Carnoy’s solution for 6-12 hours, minute lymph nodes appear milky white and the background is incompletely lysed to semitransparent flaxen fat; in such a manner, lymph nodes down to 1 mm in diameter could easily be identified. This fat clearing method can find minute lymph nodes and is helpful in accurate staging of lymph nodes: the reagents are cheap, the operation is easy, and no special instruments are needed. Our study showed that soaking tissues in Carnoy’s solution did not affect histopathological observation or immunohistochemical staining of lymph nodes.

It seems unnecessary to search further for minute lymph nodes for most breast cancer patients at late clinical stage with more lymphadenectasis. In this series, though fat clearing method found some more minute lymph nodes with metastasis in patients with stage II or stage III breast cancer, the number of these patients with lymph node metastasis did not increase. As far as accurate staging and treatment guidance are concerned, among 26 cases of minute lymph node metastasis of this series, after fat clearance 7 cases changed pN stage. For patients with tumour in early stage or without obvious lymphade- nectasis, careful and accurate detection for minute lymph nodes is necessary to avoid missed diagnosis of metastatic lymph nodes. In this group, 4 cases of stage 0 and stage I breast cancer were examined to have no metastasized lymph nodes using routine method, but after reexamination following fat clearance they were found to have minute lymph node metastasis. According to the revised cancer staging we worked out corresponding combined treatment, thus avoiding insufficient combined treatment of breast cancer

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