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Abstract

OBJECTIVE:

This study aimed to explore the positive rate of non-visualized sentinel lymph nodes (non-vSLN) [1] in breast cancer (BC) patients and the discrepancy of non-vSLN among different molecular subtypes, in order to further evaluate the clinical risk of non-vSLNs.

METHODS:

A total of 627 patients were retrospectively analyzed. These patients were pathologically confirmed with invasive breast cancer and underwent sentinel lymph node biopsy (SLNB). Various factors were compared using chi-square test. The positive rate of SLNs between non-vSLNs and visible sentinel lymph nodes (vSLNs) were compared. Moreover, factors that influenced the prognosis, such as ER, PR, HER-2, histological grade and lymph node metastasis were compared between these two groups.

RESULTS:

Among the 627 patients who underwent SLNB, 196 patients had non-vSLNs, accounting for 31.26% (196/627) and 113 patients had positive SLNs, accounting for 18.02% (113/627). Furthermore, 40.71% (46/113) of patients with positive SLNs had non-vSLN, and 17.39% (8/46) of patients with non-vSLN had HER-2+BC. In contrast, 35.82% (24/67) of patients with vSLNs had HER-2+BC. Moreover, 23.91% (11/46) of patients with non-vSLN and 5.97% (4/67) of patients with vSLNs had triple-negative breast cancer (TNBC). The metastasis rate was 41.30% (19/46) in the non-vSLN group and 43.28% (29/67) in the vSLN group. The difference in the rate of positive SLNs between the non-vSLN and the vSLN groups was statistically significant ( $P<$ 0.05), in which the positive rate of SLNs in the non-vSLN group was remarkably higher than that in the vSLN group. The differences in the proportion of HER-2+BC and TNBC between the non-vSLN and the vSLN groups were statistically significant ( $P<$ 0.05), in which HER-2+BCwas evidently higher in the vSLN group than in the non-vSLN group. Meanwhile, TNBC was markedly higher in non-vSLN group than in the vSLN group. Furthermore, differences in Luminal A subtype, Luminal B subtype and non-SLN metastasis between these two groups was not statistically significant. In addition, the difference in non-SLN metastasis rate was not statistically significant among breast cancers of different molecular subtypes and between the non-vSLN and the vSLN groups.

CONCLUSION:

Breast cancer patients with positive non-vSLNs are more likely to have a TNBC subtype relative to patients with positive vSLN. Breast cancer patients with non-vSLN have higher positive rate of SLNs. The non-SLN metastasis rate in positive SLN patients was not correlated to the molecular subtype of breast cancer.

Keywords

Breast cancer non-visible sentinel lymph node risk analysis molecular subtype

1. Introduction

Breast cancer is the leading killer that affects the female physical and mental health at present and sur-gery remains the major treatment. Minimally-invasive surgery has increasingly become the trend of surgical treatment, with the understanding that breast cancer is a systemic disease on the basis of systemic comprehensive treatment. Breast surgery, which varies from extended radical mastectomy, to modified radical mastectomy, to breast conserving surgery, to sentinel lymph node biopsy (SLNB) [2, 3] has increasingly become minimally-invasive. This not only alleviates patient sufferings and promotes rapid recovery but also reduces its impact on the quality of life of patients. We have been working on various surgical methods and safety studies of breast cancer, especially SLNB. The study on sentinel nodes guides the significance of surgery. SLNB has been studied in breast cancer to determine the value of SLN in predicting the condition of axillary nodes and its clinical significance [2]. In SLNB of breast cancer patients, is the positive rate of SLN not only related to the TNM stage of the tumor itself but also related to the molecular subtype of the tumor and other factors? In clinical practice, we find many SLN which cannot be found on imaging and we call these lymph nodes invisible sentinel lymph nodes (non-vSLN). Some of these non-vSLN biopsy patients are positive. By testing whether these certain amount of non-vSLNs are positive, can we predict if non-SLN is positive or not? Or through the detection of some positive molecules or genes related to non-vSLNs, we can predict whether the non-SLN are metastasized or not, thus avoiding ALND in patients who do not need to further ALND, even if the SLNs are positive. None of the early studies identify a positive SLNB, rather than a low-risk patient with a persistent negative SLN. At present, the report on non-vSLN is limited and the concept of non-vSLN was mentioned at the 2016 San Antonio conference [1, 3]. This study tried to analyze the factors such as non-vSLN positive and molecular subtype of breast cancer patients to explore the influencing factors on non-SLNs and evaluate the risk of non-vSLNs.

2. Material and methods

2.1 General information

A total of 627 patients treated in the Breast Department of XuZhou Tumor Hospital of Jiangsu Province and Breast Department of the First Affiliated Hospital of Anhui Medical University from January 2013 to June 2017 were retrospectively analyzed. These patients were pathologically confirmed with invasive breast cancer and underwent SLNB. Among these patients, 113 patients had positive SLNs and underwent ALND. Information on preoperative axillary lymph nodes (ALNs), as well as color Doppler ultrasound, mammography and clinical palpation were included among the collected clinical data. Meanwhile, postoperative immunohistochemical and pathological results were collected for patients with positive SLNs. All subjects of the study were female, and their ages ranged within 28–77 years old, with a median age of 48 years old. All patients with SLNB were both tumor size T1–T2 and histologic grade II-III. All patients had no neoadjuvant chemotherapy or endocrinotherapy, preoperatively.

2.2 The concept of non-vSLNs

Non-vSLN was defined as no obvious palpable lymph node enlargement on clinical palpation (palpation from two breast specialists, independently) and no ipsilateral ALN opacities on both color Doppler ultrasound and mammography.

2.3 Case selection criteria, exclusion criteria and result determination

The 627 SLNB patients and 113 positive SLNB patients were divided into two groups. Patients with non-vSLN were assigned as the non-vSLN group, while patients with vSLN (controls) were assigned as the vSLN group. Dyeing (methylene blue or nano carbon) [4] combined with fluorescence was adopted in SLNB. In order to be specific, 0.1 ml of dye was injected at the 12, 3, 6 and 9 o’clock direction of the areola, preoperatively. The dyed ALNs were searched five minutes after the injection and patients with no dyed ALNs were excluded. Next, 1–6 SLNs (median: 3) were taken out, intraoperatively. In addition, intraoperative frozen pathology was carried out and was further confirmed by postoperative paraffin pathology. Patients with over one lymph node metastasis undergoing ALND were enrolled in the present study (including micrometastases). According to 2015 St Gallen international Expert Consensus [5], the criteria for breast cancer molecular subtypes were as follows: HER-2+ was defined immunohistochemistry $+++$ or FiSH+. HER-2+ BC is HER-2+, ER-, PR-, KI67 any level. Triple-negative breast cancer (TNBC) is ER-, PR-, HER-2-, Ki67 any level. LuminaA is ER+, PR high expression, HER-2-, Ki67 lower expression. LuminaB (HER-2-) is ER+, PR lower expression/-, HER-2-, Ki67 high expression. LuminaB (HER-2+) is ER+, PR any level, HER-2+, KI67 any level. Most expert agree Ki67 $<$ 15% is lower expression, Ki67 $>$ 30 is high expression. For reflecting HER-2+BC was an independent prognostic factor [6, 7], hence, HER-2+ subtype was counted independently. HER-2+ BC and LuminaB (HER-2+) were considered as one type on the basis of breast cancer molecular subtypes,

2.4 Statistical methods

Data were analyzed using statistical software SPSS 21.0.HER-2+, triple-negative, luminal A, luminal B, vSLN and non-vSLNwere compared using chi-square test. The enumeration data were expressed in percentage (%). $P<$ 0.05 was considered statistically significant.

3. Results

3.1 Baseline data grouping

Age, menstrual status, tumor size, laterality, histological grade, as well as the ER, PR and HER-2 status of all breast cancer patients were extracted from medical records. A total of 627 patients underwent SLNB, including 113 patients with positive SLNs and 514 patients with negative SLNs. Among positive SLNs patients, 67 patients had vSLNs and 46 patients had non-vSLNs according to the preoperative axillary evaluation. Moreover, 48 patients developed non-SLNmetastasis, while 65 patients had no metastasis (Tables 1 and 2).

Table 1
Comparison of clinical data about non-vSLN and vSLN in SLN+ ( $n=$ 113)

Group Age Menstrual Tumor size Laterality

No Menopause T1 T2 Left Right

nvSLN ${}^{1}$ 48.8 $\pm$ 7.7 29 17 25 21 21 25

vSLN ${}^{2}$ 48.3 $\pm$ 8.9 43 24 39 28 27 40

$X^{2}$ 0.171 0.013 0.166 0.230

P $>$ 0.05 $>$ 0.05 $>$ 0.05 $>$ 0.05

Group HER-2 state TNBC LA LB nonSLN ${}^{3}$ Histology

$+$ $-$ $+$ $-$ $+$ $-$ $+$ $-$ $+$ $-$ II III

nvSLN 8 38 11 35 10 36 17 29 19 27 42 4

vSLN 24 43 4 63 12 55 27 40 29 38 59 8

$X^{2}$ 4.5640 7.6270 0.255 0.128 0.044 0.303

P 0.0327 0.0057 $>$ 0.05 $>$ 0.05 $>$ 0.05 $>$ 0.05

Group	Age	Menstrual	Tumor size	Laterality
		No	Menopause	T1	T2	Left	Right
nvSLN ${}^{1}$	48.8 $\pm$ 7.7	29	17	25	21	21	25
vSLN ${}^{2}$	48.3 $\pm$ 8.9	43	24	39	28	27	40
$X^{2}$	0.171	0.013	0.166	0.230
P	$>$ 0.05	$>$ 0.05	$>$ 0.05	$>$ 0.05

Group	HER-2 state	TNBC	LA	LB	nonSLN ${}^{3}$	Histology
	$+$	$-$	$+$	$-$	$+$	$-$	$+$	$-$	$+$	$-$	II	III
nvSLN	8	38	11	35	10	36	17	29	19	27	42	4
vSLN	24	43	4	63	12	55	27	40	29	38	59	8
$X^{2}$	4.5640	7.6270	0.255	0.128	0.044	0.303
P	0.0327	0.0057	$>$ 0.05	$>$ 0.05	$>$ 0.05	$>$ 0.05

1, nvSLN: non-visualized sentinel lymph nodes 2, vSLN: visualized sentinel lymph nodes. 3, Non-SLN::non- sentinel lymph nodes.

Table 2

non-SLN metastasis in different molecular types ( $n=$ 113)

Group	HER-2state		TNBC		LA		LB
	$+$	$-$	$+$	$-$	$+$	$-$	$+$	$-$
nonSLN+ ${}^{1}$	13	35	7	41	9	39	19	29
nonSLN- ${}^{2}$	19	46	8	57	13	52	25	40
$X^{2}$	0.063		0.124		0.027		0.007
P	$>$ 0.05		$>$ 0.05		$>$ 0.05		$>$ 0.05

1, non-SLN+: non-sentinel lymph nodes metastasis 2, non-SLN-: non-sentinel lymph nodes no metastasis.

3.2 Detection and comparative analysis of the different molecular subtypes between these two groups

Among the 113 breast cancer patients with positive SLNs, 11 patients in the non-vSLN group (23.91%, 11/46) and 4 patients in the vSLN group (5.97%, 4/67) had TNBC. The difference in the incidence of TNBC between these two groups was statistically significant ( $P<$ 0.05). Eight patients in the non-vSLN group and 24 patients in the vSLN group had HER-2+ with positive rates of 17.39% (8/46) and 35.82% (24/67), respectively. The difference in HER-2+ expression between these two groups was statistically significant ( $P<$ 0.05, Table 1).

Furthermore, 10 patients (21.74%, 10/46) in the non-vSLN group and 12 patients (17.91%, 12/67) in the vSLN group were luminal A type. The difference in luminal A subtypes between these two groups was not statistically significant ( $P>$ 0.05). Moreover, 36.96% (17/46) of patients in the non-vSLN group and 40.30% (27/67) of patients in the vSLN group were luminal B subtype. There were no statistically significant difference in luminal B subtype between these two groups (Table 1).

Differences in non-SLN metastasis rate among the different molecular subtypes, as well as between the vSLN and non-vSLN groups, were not statistically significant ( $P>$ 0.05, Table 2). Among the 627 patients who underwent SLNB, the positive rate of SLNs in the non-vSLN group was higher than that in the vSLN group.

4. Discussion

Currently, the determination of overtreatment and precision surgical treatment remains to be further explored. The individualized treatment for breast cancer will be more precise with the continuous development of precision medical research [8]. The aim of ALND is to understand the axillary condition, judge the tumor stage and prognosis and guide the comprehensive treatment, rather than to treat the disease. Furthermore, 20%–50% of patients undergoing ALND encounters the problem of upper extremity lymphedema [9], which maintains a treatment challenge in upper extremity lymphedema. On the basis of comprehensive treatment of breast cancer, the operation scope of surgical resection of breast cancer is gradually narrowed. Breast cancer SLNB has been clinically applied in hospitals of numerous countries. ALND is unnecessary for patients with negative SLNs, while it is necessary for those with positive SLNs [10, 11]. In particular, it was suggested in Experiment Z0011 that some breast-conserving patients who have no more than two positive sentinel lymph node (SLN) metastases can avoid ALND [12, 13, 14], provided the conditions of T $\leqslant$ 5 cm, breast conserving surgery, postoperative radiotherapy, clinically negative ALNs, and positive SLNs $\leqslant$ 2 [15]. Importantly, this has been incorporated into the National Comprehensive Cancer Network (NCCN) guidelines. Now many studies have begun to focus on how to predict non-SLN metastasis from positive SLN [16]. The purpose of these studies are to avoid excessive ALND.

But in many specialty hospitals, ALND has been conducted in all patients with positive SLNs, regardless of breast conserving treatment or mastectomy, for the principle of prudence, even though there is experimental and guideline support. TNBC and HER-2+ breast cancer patients are associated with high risk of recurrence [17]. HER-2+ breast cancer has a specific target and the DFS and OS of which can be remarkably improved by anti-HER-2 treatment [18]. No specific targeted therapy is available for TNBC, which has a poorer prognosis compared with the other subtypes [18, 19, 20]. As a result, some scholars have suggested a more aggressive surgical treatment for these patients. Gangi et al. [19] launched a prospective study that involved 3,289 breast cancer patients who underwent radical mastectomy or breast conserving surgery. The results suggested that TNBC patients did not have a higher local recurrence rate compared with other tumor subtypes, in the case of receiving breast conserving surgery. Consequently, TNBC and HER-2+ are not the relative contraindications of breast conserving surgery. The local recurrence rate in different tumor subtypes is different, which is the highest in TNBC [21]. Therefore, the possibility of non-SLN metastasis in positive SLN of TNBC and HER-2 BC is greater than that of other types? What’s the difference in SLN metastasis between Non-vSLN and vSLN and is related to molecular types? There is certain help to guide clinical work to answer these questions.

The present retrospective study analyzed the difference between non-vSLN and vSLN in patients who underwent SLNB. In comparing the positive rate of SLNs between the non-vSLN and vSLN groups, as well as among the different molecular subtypes, it was found that the positive rate of SLNs was higher in the non-vSLN group. Furthermore, the difference in non-SLN metastasis between the non-vSLN and vSLN groups was not statistically significant. The incidence of TNBC in patients with non-vSLNs was higher than in patients with vSLNs. Furthermore, the incidence of HER-2+ BC was higher in patients with vSLNs than in patients with non-vSLNs. All differences above were of statistical significance. Meanwhile, it could be concluded that non-SLN metastasis was not related to the molecular subtype. Therefore, it is not advisable to carry out ALND in patients with positive SLNs based on molecular subtype.

Therefore, non-vSLN is not a good prognostic factor. Meanwhile, the advantage of non-vSLNs on molecular subtype cannot be overestimated. Determining how to evaluate non-SLN metastasis in patients with positive SLNs? In the case of mastectomy with positive SLN, what can be done to avoid ALND, remains to be further studied.

Footnotes

Acknowledgments

We are particularly grateful to all the people who have given us help on our article.

Conflict of interest

The authors declare that they have no competing interests.

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