82

Comparison of blue dye and gammaprobe guided sentinel lymph nodebiopsy techniques in breast cancerpatients

Turkish Journal of CancerVolume 33, No.2, 2003

ABSTRACT

The introduction of sentinel lymph node (SLN) biopsy has

brought an innovative approach to the staging of early

breast cancer. Up to date two methods have been described:

a blue dye-guided lymphatic mapping procedure and a

radioactivity guided SLN biopsy technique using a hand-

held gamma probe. Aim of this study was to test the sentinel

lymph node concept which states the histopathological

analysis of sentinel lymph node is highly predictive of

metastatic involvement of other lymph nodes and to compare

gamma probe and blue dye techniques in the detection of

sentinel lymph node in breast cancer. Twenty-eight patients

with unifocal primary invasive breast cancer and clinically

negative axilla scheduled for mastectomy/lumpectomy and

ALND were included into the study. Gamma probe success-

fully identified SLN in 71% (20/28) of patients. Blue Dye

identified SLN in 61% (17/28) of patients. Either Blue dye

or gamma probe identified SLN in 79% (22/28) of patients.

The mean number of SLNs that were identified was 1.36.

Gamma probe identified more SLNs compared to blue dye

and results of statistical analysis show a significant difference

between them (Fisher’s exact chi-square test, p=0.03).

Analysis of data revealed that learning curve has a significant

effect on the success of SLN identification. The success

rate in first 6 patients was 33% (2/6) while it was 91%

(20/22) in the last 22 patients. Histopathological analysis

of SLN accurately predicted absence of the disease in the

axilla in 15 patients. In four patients there were metastatic

involvement in both SLN and other axillary lymph nodes. In

two patients micrometastasis was detected in SLN by

immunohistochemistry while non-SLNs were negative. There

was one false negative SLN biopsy, where histopathological

investigation of SLN was negative for metastases while non-

SLN were positive. We concluded that there is statistically

significant difference between blue dye and gamma probe

techniques for identifying SLN and gamma probe identifies

more SLN compared to blue dye. However, they are com-

plementary techniques and combined method works better.

This study also supports the clinical validity of SLN biopsy

in breast cancer and confirms that histopathological analysis

of sentinel lymph node is highly predictive of metastatic

involvement of other non-SLNs in axilla. [Turk J Cancer

2003;33(2):82-90]

KEY WORDS:

Sentinel lymph node, breast cancer, lymphoscintigraphy,

gamma probe, blue dye

INTRODUCTION

Routine axillary node dissection is currently the standard

of care in clinically node negative breast cancer patients.

ÖMER UGUR1, M. FANI BOZKURT1, ISKENDER SAYEK2, GÖKHAN GEDIKOGLU3, ATAÇ BAYKAL2,

ERHAN HAMALOGLU2, ILKER ETIKAN4, ALI KONAN2, BELKIS ERBAS1

Departments of 1Nuclear Medicine, 2Surgery, 3Pathology and 4Biostatistics, Hacettepe University Faculty of Medicine, Ankara-Turkey

83Ugur et a l .

Axillary lymph node dissection (ALND) has a well estab-

lished role in regional disease control and it provides

information about histopathological status of axillary lymph

nodes which has significant prognostic and adjuvant ther-

apeutic implications (1,2). However, 70-80% of clinically

node negative patients prove to be histopathologically node

negative and they must still sustain the expense and mor-

bidity of this procedure including complications such as

neuropathies, seromas, shoulder movement restriction and

upper extremity lymphedema in 5-8% (3-5). After the

widespread use of screening mammography, patients with

breast cancer now present with smaller tumors than in the

past and as a result the incidence of axillary nodal involve-

ment is lower than before (6). There is a tendency to use

breast conserving surgery without compromising local

control or long term survival. Sentinel lymph node (SLN)

biopsy is a recently developed minimally invasive technique

for staging the axilla in patients with breast cancer (7). SLN

is the first lymph node in a given lymphatic basin to receive

lymphatic flow from a primary tumor site. This technique

introduces a new concept, namely that of selective lym-

phadenectomy in breast cancer on the basis of the histolog-

ical status of the SLN and avoiding unnecessary ALND

(8).

Moreover, relapse rates of up to 30% in node negative

breast cancer patients with single section hematoxylin and

eosin (H&E) histopathological examination gives out

skepticism about the routine histopathological examination

of ALND specimen (9). Detailed histopathological investi-

gation of the SLN including multiple sections and immu-

nohistochemistry for micrometastases gives us the oppor-

tunity to accurately predict the status of the distant lymph-

node basin (10). However, the optimal technique for SLN

biopsy is still debated. Initial studies to locate sentinel

lymph node were conducted with vital blue dyes with less

favorable results (11,8). Though, for this technique to

become widely accepted in the management of breast cancer,

it needs to identify the SLN reliably. Recently, intraoperative

gamma probe was used for this purpose. Aim of this study

was to test the sentinel lymph node concept which states

the histopathological analysis of sentinel lymph node is

highly predictive of metastatic involvement of other lymph

nodes and to compare gamma probe and blue dye techniques

in the detection of sentinel lymph node in breast cancer.

MATERIALS AND METHODS

Patients and study protocol

Twenty-eight patients with unifocal primary invasive

breast cancer with clinically negative axilla scheduled for

mastectomy/lumpectomy and ALND were included into

the study. Patients with multicentric primary breast cancer

or clinically positive regional lymph nodes were excluded.

The study protocol was approved by the Hacettepe Univer-

sity Institutional Review Board, and prior written informed

consent was obtained from all patients.

Radiopharmaceutical injection technique

and lymphoscintigraphy

2-12 hours before operation 500-2500 uCi of either Tc-

99m-colloidal rhenium sulphide (Nanocolloid, CIS bioint-

ernational, France) or Tc-99m-colloidal tin (Amerscan

Hepatate II Agent, Nycomed Amersham plc, UK) was

injected intradermally at the same quadrant with the tumor.

Immediately following injection, dynamic lymphoscintig-

raphy was performed using gamma camera (e-cam, Siemens,

Erlangen, Germany) equipped with a high resolution, low-

energy, parallel hole collimator. Dynamic 64x64 matrix

images were recorded every minute for 30 min. After

dynamic imaging 5 min static images of anterior and lateral

breast were acquired at 256x256 matrix (Figure 1). In

addition, Co-57 flood source transmission imaging was

used to delineate body contours. Any radioactivity accumu-

lation suggestive of SLN was marked on the skin with the

help of Co-57 tipped pencil marker.

Fig 1. Lymphoscintigraphy revealed SLN in the axilla (arrow:SLN, arrow head: injection site)

84 Sent inel Node Biopsy and Breast Cancer

Gamma detection probe

Intraoperative radioactive SLN localization was per-

formed using a gamma photon detection probe (Neoprobe

2000, Neoprobe corporation, Dublin, Ohio, U.S.A) with

energy peak set at 140 keV-20% window.

Blue dye

Injectable sterile solutions of 1% isosulphan blue (mono-

sodium salt of 2,5-disulphonated triphenyl methane) was

prepared by the Department of Pharmaceutical Technology

at Hacettepe University Faculty of Pharmacy using a stock

solution obtained from Sigma company (5 g isosulphan

blue, Sigma catalog no: P1888, Sigma-Aldrich chemical

co., Deisenhofen, Germany).

Intraoperative lymphatic mapping and

sentinel lymph node biopsy

The gamma probe was ensheathed in a sterile endoscopic

probe cover and a sterile tape was tied over the end of

probe. After the induction of general anesthesia, 5 ml 1%

Isosulphan Blue was injected and gentle massage was

applied for a few minutes. Following the skin preparation

and draping of the operative field a 10 sec probe count was

taken from the sternal notch as a background count. The

pencil mark on the axilla correspondent to the SLN was

counted and then intramammary, supraclavicular, infraclav-

icular, parasternal and rectus sheath regions were surveyed

with gamma probe methodically in a grid pattern over the

skin. Cutaneous hot spots were delineated and carefully

marked; these signify the accumulation of radioactivity in

one or more SLN. SLN biopsy was performed through a

standard anteroposterior incision placed in the lower axillary

hair-bearing skin, as routinely performed in formal axillary

lymphadenectomy. The gamma probe counts guided the

dissection and any radioactive node(s) that have in-vivo

counts of at least 3 times more than background counts

were excised. Ex-vivo counts from the excised node(s)

were recorded. The lymph node(s) with in-vivo counts of

at least 3 times and with ex-vivo counts of at least 10 times

that of the background was accepted as SLN(s). After

excision of the SLN(s), mastectomy was performed by

developing mastectomy flaps. Routine axillary lymph node

dissection through levels 1, 2 and/or 3 was then completed.

The SLN(s), mastectomy and axillary lymph node dissection

material were kept as separate specimen for the pathological

examination (Figure 2).

Pathologic evaluation

Histopathologic analysis was

performed by detailed micro-

scopic examination of three sec-

tions from each bivalved SLN and

one section from non-SLN. In

addition to H&E staining cytok-

eratin and epithelial membrane

antigen (EMA) immunohis-

tochemistry was also applied to

SLN.

Statistical analysis

The significance of the dif-

ference between gamma probe

and blue dye for SLN detection

were made by Fisher’s exact chi-

square test.Fig 2. Gamma probe (A). Gamma probe detection of SLN (B). Blue dye injection (C). Excisionof blue node (arrow) (D)

85Ugur et a l .

RESULTS

Tumor characteristics

Histopathological diagnosis and location of the primary

tumor were given in table 1. Twenty patients had infiltrating

ductal carcinoma. Primary tumor stage was T1 in 16 patients,

T2 in 6 patients, and T3 in 3 patients.

Comparison of blue dye and gamma probe

technical success in localising SLN

Gamma probe successfully identified SLN in 71%

(20/28) of patients. Blue dye identified SLN in 61% (17/28)

of patients. Either Blue dye or gamma probe identified

SLN in 79% (22/28) of patients. The mean number of SLNs

that were identified was 1.36.

Table 1

Histopathological diagnosis, primary tumor size and location

Patient no. Histopathological Primary tumor Gamma Blue Number Location of

(Age/sex) Diagnosis size (cm) Probe Dye of SLN primary tumor

1. (SH) (74/F) Infiltrative ductal carcinoma 6 - - - UOQ

2. (SK) (45/F) Infiltrative ductal carcinoma NA + + 1 LOQ

3. (ME) (53/F) Infiltrative ductal carcinoma 1.2 + + 2 LOQ

4. (AK) (55/F) Infiltrative ductal carcinoma 1.1 - - - UOQ

5. (FK) (49/F) Mixed infiltrative ductal-lobular 3.5 - - - LOQ

carcinoma

6. (AD) (57/F) Infiltrative ductal carcinoma 0.3 - - - Central

7. (SP) (46/F) Infiltrative ductal carcinoma 5 + + 1 UOQ

8. (YÜ) (68/F) Microinvasive ductal carcinoma 0.5 + + 1 UOQ

9. (CG) (25/F) Mucinous carcinoma 6 + + 1 UOQ

10. (AÇ) (53/F) Infiltrative lobular carcinoma 2.5 - - - Outer central

11. (HA) (46/F) Infiltrative ductal carcinoma 1.2 + - 1 UIQ

12. (MA) (51/F) Microinvasive ductal carcinoma 0.8 + - 1 UIQ

13. (SS) (40/F) Infiltrative ductal carcinoma 1.7 + - 2 UOQ

14. (EFS) (62/F) Infiltrative ductal carcinoma 3 + + 1 UOQ

15. (SS) (59/F) Intraductal carcinoma NA + + 2 LOQ

16. (NG) (50/F) Mixed infiltrative ductal-lobular 2 - + 1 Central

carcinoma

17. (LT) (57/F) Mixed infiltrative ductal-lobular 1.5 + + 2 Bilateral UOQ

carcinoma

18. (AS) (46/F) Infiltrative ductal carcinoma 1.8 + - 2 Upper central

19. (HÇ) (51/F) Infiltrative ductal carcinoma 3 + + 1 Outer central

20. (RC) (44/M) Intraductal carcinoma 2.5 + + 1 UOQ

21. (NC) (49/F) Ductal carcinoma in situ NA + + 1 UOQ

22. (SY) (44/F) Infiltrative ductal carcinoma 1.8 + + 3 UOQ

23. (SS) (76/F) Infiltrative ductal carcinoma 1.7 + + 1 LOQ

24. (SK) (48/F) Ductal carcinoma in situ <2 mm - + 1 LOQ

25. (HÇ) (49/F) Infiltrative ductal carcinoma 1.6 + + 1 UOQ

26. (ST) (57/F) Ductal carcinoma in situ 0.1 - - - Outer central

27. (FÇ) (58/F) Infiltrative ductal carcinoma 1.3 + - 2 UOQ

28. (FK) (57/F) Infiltrative ductal carcinoma 1.5 + + 1 UOQ

UOQ: Upper outer quadrant, LOQ: Lower outer quadrant, UIQ: Upper inner quadrant, NA: Primary tumor size is not

available because pathologic examination of the primary tumor was undertaken by another institute

86 Sent inel Node Biopsy and Breast Cancer

In a patient (patient no: 5) whom there was extensive

local and axillary tumoral involvement, gamma probe

identified a hot spot in internal mammary region but neither

gamma probe nor blue dye was capable of identifying SLN

in axillary region probably due to complete infiltration of

the nodes with tumor and leaving no lymphoid parenchyma

to localize radiocolloid and blue dye in axilla (Figure 3).

We did not explore internal mammary area to identify hot

spot and accepted this case as directional lymphatic flow

change due to mechanical obstruction. In another patient

(patient no: 8) lymphoscintigraphy showed a dim lymphatic

truct with an activity 4x background with in-vivo gamma

probe. Although counts were less than expected, it was

accepted as a sentinel lymph node. However blue dye could

not identify any SLN intraoperatively. Histopathological

examination demonstrates widespread fatty degeneration

in the lymph node with decreased functional parenchyma

for radiocolloid uptake (Figure 4). In other 4 patients with

unsuccessful localization no obvious reason for failure can

be identified but these patients were within the first 6

patients and inexperience of the team can be a reason for

the failure. Comparison of blue dye and gamma probe in

identifying sentinel lymph node is tabulated in table 2.

Gamma probe identified more SLNs compared to blue dye

and results of statistical analysis shows a significant differ-

ence between them (Fisher’s exact chi-square test, p=0.03).

Fig 3a. Diffuse infiltrative metastasis in sentinel lymph node(H&E, x30) (patient no:5)

Fig 3b. High power view from diffuse infiltrative metastasisin sentinel lymph node (x115)

Fig 4. Lipomatous infiltration in sentinel lymph node (H&E,x30)(Patient no:8)

87

Effect of learning curve on the success

of SLN identification

Analysis of data revealed that learning curve has a

significant effect on the success of SLN identification. The

success rate in first 6 patients was 33% (2/6) while it was

91% (20/22) in the last 22 patients.

Correlation with histopathologic status

of SLN and axillary involvement

Histopathological analysis of SLN accurately predicted

absence of the disease in the axilla in 15 patients. In 4

patients there were metastatic involvement in both SLN

and other axillary lymph nodes. In two patients microme-

tastasis was detected in SLN by immunohistochemistry

while other axillary lymph nodes were negative (Figure 5).

There was one false negative SLN biopsy, where histopatho-

logical investigation of SLN was negative for metastases

while non-SLN were positive (patient no: 25).

Toxicity and adverse effects

There were no allergic or idiosyncratic events related

to the use of Tc-99m labeled colloid lymphoscintigraphic

agents and blue dye.

Table 2

Comparison of the gamma probe and blue dye for the localization of the SLN*

Blue Dye

Positive Negative Total

Gamma Probe

Positive 15 (75%) 5 (25%) 20 (100%)

Negative 2 (25%) 6 (75%) 8 (100%)

Total 17 (60.7%) 11 (39.3%) 28 (100%)

* Gamma probe identified more SLN compared to blue dye and this is statistically significant

(Fisher’s exact chi-square test p=0.03)

Fig 5a. Micrometastasis in sentinel lymph node (arrow) (H&E,x30) patient no.18)

Ugur et a l .

88 Sent inel Node Biopsy and Breast Cancer

Both methods have merits and disadvantages (12). Using

blue dye technique surgeon can visualize and follow lym-

phatic truct to dissect SLN. Moreover, blue dye is widely

available and simple to use. However, timing of the blue

dye is crucial for the success of the procedure. If it is

injected too early there would be extensive blue staining

in the nodal basin making the dissection of SLN very

difficult. On the other hand if the injection is administered

too late, successful localization may fail owing to the

inability of the dye to reach the sentinel node because of

disruption of lymphatic channels during dissection. Pre-

operative lymphoscintigraphy and intraoperative gamma

probe techniques give us the knowledge about the location

of SLN before the surgery. Another important advantage

of probe guided surgery is that complete excision of the

SLNs can be verified by directing the probe into the incision

to measure residual activity. Moreover, gamma probe will

give the surgeon a sense of direction and allows detection

of non-visible nodes due to their radioactive content. There

DISCUSSION

The introduction of SLN biopsy has brought an innova-

tive approach to the staging of early breast cancer. Up to

date two methods have been described: a blue dye-guided

lymphatic mapping procedure and a radioactivity guided

SLN biopsy technique using a hand-held gamma probe.

Fig 5b. EMA (+) micrometastasis (Strept ABC, x30)

Fig 5d. High power view from EMA (+) micrometastasis (Strept

ABC, x115)

Fig 5c. High power view from micrometastasis (H&E, x115)

89

is increased evidence in the literature to support better

results when both detection methods are combined, com-

pared with the use of these techniques alone (13). Cserni

and associates (14) reported that combined technique has

advantages like higher identification rate, higher accuracy

level and a lower false negative rate.

In the present study, we present Hacettepe University

Medical Center experience with dye-guided and dye plus

gamma probe guided SLN biopsy and compared these two

methods. As demonstrated in earlier studies on SLN biopsy

in breast cancer, we experienced a learning period (14,15).

In the first 6 patients our success rate to identify SLN was

only 33%, compared to 91% in the last 22 patients. We

used intradermal radiocolloid injection technique in our

study as suggested to be a superior technique compared to

peritumoral injection and this may result our high success

rate at the end of the learning curve (12).

It has also been suggested that false negative rate (SLN

histopathology (-) for metastases, non SLNs histopathology

(+) for metastases) is higher during the introductory phase

(16). This is not validated by our results that we observed

only one false negative case. As the number of patients in

our series increased we may observe increase in false

negative rate. In order to SLN biopsy to replace axillary

lymphadenectomy false negative rate should be low. Meta-

analysis of the data from 685 patients who had SLN biopsy

for breast cancer shows that the false negative rate ranges

between 5.1% and 7.6% (6). Several factors may account

for the increased false negative rate in SLN biopsy such

as previous surgery, multifocality of tumor and extensive

lymphovascular invasion. Previous surgery may explain

the false negative case in our series. Another reason for

false negative rate in blue dye only method is that surgeon

may be satisfied with the number of SLNs identified and

only some of these may be true SLNs. However, in the

combined method surgeon can detect non-visible SLNs

due to their radioactive content and this may explain our

low false negative rate. The mean number of SLN identified

in our study (1.36) and the number of both blue and radio-

active nodes (53%) are in concordance with the larger

series in the literature (17,18). Although gamma probe

identified more SLNs compared to blue dye, statistical

analysis could not find a significant difference between

them. Lymphatic flow change due to mechanical obstruction

and fatty degeneration in the lymph node are responsible

for 2 of the 6 cases which both methods are unsuccessful

in localising SLN. These are also common reasons reported

in the literature for failure (12). We also did not observe

lymphatic drainage occurring to lymph nodes outside the

axilla which is a less common finding with intradermal

technique compared to peritumoral injection such as the

lymphatic drainage which Uren and associates (19) observed

occurring to lymph nodes outside the axilla including the

internal mammary in more than half of the patients with

peritumoral injection technique (6). In two patients mi-

crometastases in SLN were detected using cytokeratin and

EMA immunohistochemistry. At present, there are conflict-

ing data on the prognostic significance of detecting mi-

crometastatic disease in the SLN by immunohistochemistry,

how this information could be used in clinical practice is

unclear (6). Should these patients be subjected to toxicity

of adjuvant chemotherapy or further radiotherapy of axilla?

However, there is evidence that sensitive detection of SLN

micrometastases, enhanced by paraffin step sections and

cytokeratin immunohistochemistry is important because a

significant number of patients with SLN micrometastases

have additional non-SLN metastases, and further surgery

and adjuvant therapies may be warranted. However, the

prognostic significance of a few cytokeratin-positive cells

remains to be determined (20).

We concluded that there is statistically significant

difference between blue dye and gamma probe techniques

for identifying SLN and gamma probe identifies more SLN

compared to blue dye. However, they are complementary

techniques and combined method works better. This study

also supports the clinical validity of SLN biopsy in breast

cancer and confirms that histopathological analysis of

sentinel lymph node is highly predictive of metastatic

involvement of other non-SLNs in axilla.

ACKNOWLEDGEMENT

This work was supported in part by the Hacettepe

University Research Fund no: 00.01.101.011.

Ugur et a l .

90 Sent inel Node Biopsy and Breast Cancer

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