Sentinel lymph node biopsy with carbon nanoparticle suspension after neoadjuvant chemotherapy for breast cancer patients

Abstract

Introduction

The aim of the study was to explore the feasibility of performing sentinel lymph node biopsy (SLNB) using a carbon nanoparticle suspension (CNPS) after neoadjuvant chemotherapy in breast cancer patients.

Methods

Some 152 patients diagnosed with primary breast cancer (cT1-3N0-2M0) were recruited. Patients were divided into two groups according to axillary lymph node (ALN) status after four to six cycles of neoadjuvant chemotherapy. All patients received a CNPS injection, after which SLNB and axillary lymph node dissection (ALND) were performed.

Results

Sentinel lymph nodes (SLN) of 143 patients were identified; with an accuracy rate of 94.4% and a false-negative rate of 9.9%. Group A included 67 patients, and the detection, accuracy and false-negative rates within this group were 95.5%, 96.9% and 6.7%, respectively. The corresponding rates for group B (85 patients) were 92.9%, 92.4% and 11.8%, respectively.

Conclusions

CNPS is an ideal tracer for improving the detection rate of SLN and can be used to determine SLN status following neoadjuvant chemotherapy.

Introduction

Breast cancer is the most common type of cancer in women.¹ Axillary lymph nodes (ALNs) are the main channels for lymphatic drainage of the breast, and the status of the ALNs plays a significant role in the treatment decisions and prognosis of breast cancer patients. Axillary lymph node dissection (ALND) is the most accurate method for assessing breast cancer stage. However, ALND can cause a wide range of complications, such as postoperative lymphoedema, shoulder dysfunction, seroma, skin numbness and chronic pain. Sentinel lymph nodes (SLNs) are the first location for a specific primary tumour to metastasise; if the SLN has not been invaded, metastasis of other lymph nodes or organs is less likely to have occurred.^2,3 The results of multiple prospective randomised trials, such as SNB185, NSABPB232 and ALMANAC, all confirmed that patients receiving sentinel lymph node biopsy (SLNB) had the same degree of positive ALNs as those assessed by ALND, and that SLNB could provide accurate ALN staging; if no SLN metastasis was apparent, ALND was avoidable and morbidity reduced. Moreover, SLNB can shorten surgery duration and reduce morbidity. Therefore, the American Society of Clinical Oncology (ASCO), the National Comprehensive Cancer Network (NCCN), the St. Gallen Consensus, and other expert consensuses for breast cancer recommend SLNB as the gold standard of axillary management for ALN-negative patients.⁴

Currently, the primary methods of determining SLN status include the dye method, the radionuclide detection method or a combination of these. Dye is widely used in the clinic due to its desirable tracer effect, convenience and the fact that no specialised equipment is required, or radioactive contamination occurs. Carbon nanoparticles (CNPs) are third-generation dye tracers that are derived from nanotechnology. With a uniform diameter of approximately 150nm, CNPs are larger than capillaries, and therefore preferentially enter the lymph nodes through lymphatic vessels (rather than blood vessels), staining the lymph nodes black. In addition, CNPs exhibit no mutagenicity or carcinogenicity and have only minimal side-effects. Therefore, CNP tracers can be used as an aid to SLNB during breast cancer surgery. However, the value of CNPs in determining SLN status has yet to be defined.⁵

Neoadjuvant chemotherapy (NAC) plays an important role in breast cancer treatment by downsizing the primary tumour. It can simplify subsequent surgery, eg allow breast conservation or transform an inoperable tumour into an operable one. NAC is currently the standard mode of treatment for patients with locally advanced breast cancer.⁶ However, whether SLNB can accurately assess the ALN status of patients following NAC remains to be confirmed. Currently, the available literature does not fully support this hypothesis, with several studies presenting controversial results. Therefore, further investigation is required to address this issue. The aim of the present study was to explore the feasibility of using a carbon nanoparticle suspension (CNPS) to determine SLN status, and thus replace ALND following NAC in breast cancer patients.

Methods

Patients

A total of 152 patients with primary invasive breast cancer were enrolled in the study; the median patient age was 49 years (range 29–68; Table 1) and patients with stage cT1-3N0-2M0, Karnofsky Performance Scale scores above 90 and no surgical contraindication were included. All patients received four to six cycles of NAC before surgery, and the status of the ALN was assessed by physical examination, ultrasound and mammography. For some patients, the ALN status was confirmed by core biopsy or fine-needle aspiration (FNA), depending on the diameter of the node. If the node was palpable (>2cm) and superficial, core biopsy was used, otherwise, FNA was used. Patients with a previous history of axillary surgery or of endocrine or radiation therapy for breast cancer were excluded from the study. Patients with pregnancy-associated or inflammatory breast cancer, or those with multiple lesions were also excluded. Patients were divided into two groups according to the characteristics of their breast cancer: group A were ALN-negative (cN0) before and after NAC; group B were ALN-positive (cN1-2) before, but ALN-negative (ycN0) after NAC.

Table 1 .

Clinicopathological characteristics of eligible patients (n = 152)

Characteristic	Subgroup	n (%)
T-stage	T1	57 (37.5)
	T2	60 (39.5)
	T3	35 (23.0)
N-stage before chemotherapy	N0	67 (44.1)
	N1	55 (36.2)
	N2	30 (19.7)
Age	>50	62 (40.8)
	<50	90 (59.2)
Primary tumour site	Outer upper quadrant	83 (54.6)
	Lower outer quadrant	69 (45.4)
Receptor status	ER+/PR+	98 (64.4)
	ER+/PR–	12 (7.9)
	ER–/PR+	0 (0.0)
	HER2/neu+	35 (23.0)
	HER2/neu–	117 (77.0)

T = tumour; N = node; ER = oestrogen receptor; PR = progesterone receptor; HER2 = human epidermal growth factor receptor 2

Sentinel lymph node biopsy

Primary tumour removal, SLN dissection and ALND were performed in all patients. 0.5ml CNPS was administered with a 1ml disposable syringe 10–30min prior to surgery; the CNPS was injected into the subcutaneous area of the gland around the tumour or areola, 0.08ml/injection up to a total of 0.4–0.5ml. The injection site was gently massaged for 3–4min to allow full diffusion. A section cut was taken at the junction between the lateral margins of the pectoralis major and the axilla. The stained lymphatics and SLNs were subsequently located, and the SLNs were excised prior to ALND. The status of the lymph nodes was determined by embedding the tissues in paraffin for subsequent histopathological examination.

Statistical analysis

The median patient follow-up period was 2 years. SPSS v. 22 (IBM Corp., Armonk, NY, USA) was used for data analysis, and the χ² test was performed to analyse the detection, accuracy and false-negative rates. The detection rate was defined as the percentage of patients whose SLN was successfully detected. The accuracy rate was defined as the percentage of patients with consistent results for SLN and ALND examinations, and the false-negative rate was defined as the percentage of SLN-negative, but ALND-positive patients. A value of p < 0.05 was considered to indicate a statistically significant difference.

Results

Sentinel lymph node detection rate and false-negative rate

Among the 152 patients who received SLNB following NAC, the SLNs of 143 cases were successfully detected using CNPS (Figure 1). The average number of nodes detected during ALND was 15.4. According to the evaluation standard of the SLN technique of the University of Louisville, the detection rate of SLN in the present study was 94.1% [95% confidence interval (CI) 88.3–95.2]. Eight SLN-negative patients were confirmed to also be ALN-positive, with an accuracy rate of 94.4% (95% CI 86.3–96.4) and a false-negative rate of 9.9% (95% CI 4.6–14.5; Table 2). Group A included 67 patients, 64 of whom were identified as SLN-positive with a detection rate of 95.5% (95% CI 84.0–97.2); two SLN-negative patients were confirmed to be ALN-positive, with an accuracy rate of 96.9% (95% CI 87.8–98.5) and a false-negative rate of 6.7% (95% CI 3.6–12.1). The corresponding rates for group B (85 patients) were 92.9% (95% CI 85.6–95.8), 92.4% (95% CI 81.7–96.0) and 11.8 (95% CI 5.9–17.4), respectively. The false-negative rate for group B was 26.7% (95% CI 18.2–31.6) in patients in whom one positive SLN was detected, 12.5% (95% CI 8.5–16.9) in patients in whom two SLNs were detected, and 0% (0 of 13) in patients in whom more than two SLNs were detected (Table 3).

Figure 1 .

A carbon nanoparticle suspension (CNPS; 0.5ml) was administered with a 1ml disposable syringe 10–30 min before surgery. The injection site was gently massaged for 3–4 min to allow the CNPS to diffuse. A section was cut from the junction between the lateral margin of the pectoralis major and the lateral margin of the axilla. The stained lymphatics and sentinel lymph nodes were subsequently located and are displayed in the representative image. CNPS = carbon nanoparticle suspension.

Table 2 .

Histopathological results of SLNB and ALND in all patients

ALN (n)	SLN (n)		Total (n)
	Positive	Negative
Positive	45	8	53
Negative	28	62	90
Total	73	70	143

SLNB = sentinel lymph node biopsy; ALND = axillary lymph node dissection; ALN = axillary lymph node; SLN = sentinel lymph node

Table 3 .

Histopathological results of SLNB and ALND in group B

ALN (n)	SLN detected (n)						Total (n)
	1		2		>2
	Positive	Negative	Positive	Negative	Positive	Negative
Positive	10	4	7	2	9	0	32
Negative	1	4	7	13	4	18	47
Total	11	8	14	15	13	18	79

ALN = axillary lymph node; SLN = sentinel lymph node

Influence factors for SLN detection and false-negative rate

The histopathological results of 152 patients (groups A and B) were analysed according to tumour size, age, ALN status before chemotherapy, primary tumour and CNPS injection site, and the results are shown in Table 4. All 57 T1 stage breast cancer patients were SNL-positive, and only one case was proven to be false-negative. Among the remaining 95 patients (with T2–3 stage breast cancer), SLNs were detected successfully in 86 cases, not detected in nine cases, and seven cases were false-negative Risk Ratio [(RR) = 4.2]. In addition, the SNLs of 64 of 67 patients with N0 stage disease were detected successfully, with two false-negative cases. Accordingly, the SNLs of 79 of 85 patients with N1–2 stage breast cancer were identified successfully, with six false-negative cases (RR = 2.4). There were no significant effects from other factors, including age, primary tumour site and CNPS injection site.

Table 4 .

Factors influencing the detection and false-negative rates of SLNs

	Subgroup	Patients (n)	SLN success (n)*	SLN failure (n)**	False- negative cases (n)	Risk ratio
T Stage	T1	57	57	0	1	4.2
	T2–3	95	86	9	7
N stage before chemotherapy	N0	67	64	3	2	2.4
	N1–2	85	79	6	6
Age	>50	62	60	2	3	1.1
	<50	90	83	7	5
Primary tumour site	Outer upper quadrant	83	78	5	4	1.2
	Lower outer quadrant	69	65	4	4
Injection site	Around the tumour	46	44	2	2	1.3
	Areola	106	99	7	6

SLN = sentinel lymph node; T = tumour; N = node

*Number of patients whose SLNs were detected successfully

**Number of patients whose SLNs were not detected successfully

Follow-up

After 2 years of follow-up, no distant metastasis or local recurrence occurred in any of the patients, and no patients died. Only four of the 152 patients experienced numbness of the upper limbs, and one patient exhibited upper limb oedema. No abnormalities of the liver or kidney were reported in any of the patients.

Discussion

The ALN serves an important role in breast cancer staging, adjuvant treatment selection and the prognosis of patients with breast cancer. SLNB is a standard strategy for ALN staging in patients with early-stage breast cancer, and can accurately predict the occurrence of ALN metastasis:⁷ this prevents the need for ALND in patients with early-stage ALN-negative breast cancer, effectively reduces the incidence of postoperative complications, protects the function of the affected upper limb and also improves patient quality of life.⁸ However, the current recommendations do not offer precise guidance on whether SLNB is a suitable means of ALN assessment in patients initially presenting with ALN-positive, but that revert to ALN-negative breast cancer following NAC. In the present study, SLNB was conducted in breast cancer patients after NAC using CNPS as a tracer. The detection, accuracy and false-negative rates of SLNB were 94.1%, 94.4% and 9.9%, respectively, and meet current standards set by the NCCN in 2015, requiring a false-negative rate below 10%.⁹

The results of the present study confirmed that use of CNPS as an enhancement to SLNB was reliable and accurate in breast cancer patients following NAC, although in 2005, ASCO indicated that SLNB was not recommended after NAC.¹⁰ From an anatomical perspective, when carried out after NAC, SLNB can result in destruction of the lymph node reflux network, leading to lymphatic fibrosis and obstruction, as well as changes in the lymphatic drainage channels, which may affect SLN detection during surgery.¹¹ However, with advances in medicine, and based on a great deal of clinical evidence, attendees of the 2009 St. Gallen meeting supported an opposing view.¹² Recently, two meta-analyses have shown that after NAC, the detection and sensitivity rates of SLNB were above 90% and 88% respectively, suggesting that SLNB is an accurate and feasible method of detection in ALN-negative breast cancer patients who have received NAC.^13,14

In the present study, comparative analysis revealed that SLNB after NAC could be affected by four factors: tumour size, N-stage before chemotherapy, tumour site, and patient age. Also, the false-negative rates of patients with T1 and T2 stage breast cancer were significantly different (RR = 4.2). It is generally believed that the larger the tumour, the more obvious the interference with lymphatic drainage, and the detection rate of SLNB will decrease as tumour size increases.¹⁵ Metastatic cells can impair lymphatic drainage, which can affect aggregation of the tracer in the metastatic lymph nodes; lymph nodes without metastasis exhibit tracer aggregation, affecting the detection rate and thus causing false-negative results.¹⁶ It has been reported that SLNB accuracy can reach 100% when the tumour volume decreases to 1.6cm or less, without false-negative results.¹⁷ In the present study, there were no significant differences in SLN detection rate between N0 and N1–2 stage patients before NAC. However, the false-negative rate was significantly different between the two groups (RR = 2.4). The SLNB accuracy rate was 92.4%, and the false-negative rate was 11.8% in ALN-positive patients (N1–2) before NAC. However, the false-negative rate was 0% (0 of 13) when more than two SLNs were detected in these patients, which was consistent with the results of the ACOSOGZ1071 trial and the SNFNAC study, first reported at the 2013 ASCO annual meeting.^16,17 These results indicate that if patients with N1–2 stage tumours revert to ALN-negative status following NAC, SLNB may be used when more than two SLNs are detected.

Tumour site and patient age may also affect the detection of SLNB.¹⁸ The internal mammary nodes (IMNs; also known as the parasternal nodes) are a network of lymph nodes in the inner breast, and another important route by which tumours may metastasise. However, because of the deep location and inconvenient detection of IMNs, they are often ignored as ‘hidden’ SLNs. Studies have confirmed that in some ALN-negative patients, IMN was identified as positive.^19,20 Therefore, IMN may need to be taken into consideration in some cases, eg the tumour is in a medial or central breast location.

Finally, in older patients, replacement of lymphatic tissue with adipose tissue can lead to decreased phagocytosis and mechanical barrier capacity of reticuloendothelial cells in lymph nodes, reducing tracer excretion time, thus increasing the false-negative rate of SLNB.²¹ However, we did not see the significant effects of age in our study (RR = 1.1), which may have been the result of a small sample size.

At present, SLN tracers can be broadly categorised into three types: blue dye, radionuclides and a combination of both. For SLNB, higher tracer sensitivity generates improved effects, higher detection rate and lower false-negative rates. Studies have shown that the combination of blue dye and radionuclides can improve the detection rate of SLNB by 1.3% and reduce the false-negative rate by 2.5%.^22,23 However, although the radionuclide detection method is accurate and reliable, the preoperative preparation process is complex and expensive, and the correct protection of radioactive isotopes is difficult to achieve in small-scale hospitals.

CNPS is a third-generation dye tracer. Carbon particles of uniform size are produced by nanotechnology and possess strong lymphatic specificity and an ideal tracer effect. With a diameter of approximately 150nm, CNPs cannot penetrate the blood vessels, which improves trace specificity. In addition, animal experiments have shown that CNPs have the advantage of being neither mutagenic nor carcinogenic, and do not adversely affect the central nervous, cardiovascular or respiratory systems. Therefore, CNPS is convenient and safe to use during SLNB.⁵

In the present study, the detection rate of SLN using CNP was 96.9%, with a sensitivity and accuracy rate of 91.2% and 94.8%, respectively, as well as a false-negative rate of 8.8%, which accurately reflects the status of the ALN. Moreover, with strong specificity and long staining time, CNPs can ALN dissection, which can be clearly displayed in subsequent ALN management.

Conclusions

In conclusion, the results of our study indicate that CNPS is an ideal SLNB tracer for breast cancer after NAC.