Impact of breast cancer molecular subtypes on the incidence of axilllary lymhp nodes metastases Emad Sadaka1

Impact of breast cancer molecular subtypes on the incidence of axilllary lymhp nodes metastases
Emad Sadaka1&Walid Almorsy2
Clinical Oncology Department, Faculty of Medicine, Kafer Elsheikh university1, Tanta university2
Purpose: Breast cancer has at least four molecular subtypes, Luminal A; Luminal B, HER2+ and triple negative with significant differences in prognosis and ALN involvement.This study aimed to investigate the impact of breast cancer molecular subtypes on the incidence of axillary lymph nodes metastases Methods: Three hundred and twenty nine female patients with invasive breast cancer were included in this study. Age at diagnosis, menopausal status, tumor size, histological type and grade, lymph node status and molecular subtypes were recorded.Four major molecular subtypes were classified, Luminal A; Luminal B, HER2+ and triple negative. Results: The mean age was 51.34 years old.Most of patients (86.9%) have Invasive ductal carcinoma. One hundred sixty four (49.5%) patients had node negative disease while 166 (50.5%) had node positive disease. Luminal A molecular subtype was recorded in 87 patients (26.4%), luminal B 163 patients (49.5%), HER-2+ 35 patients (10.6%) and TNBC 44 patients (13.5%). There are differences in ALN positivity by molecular subtypes, node positive disease was (11.5%) among luminal A patients, luminal B patients (66.9%), HER2+ (91.4%) and TNBC (34.1%). There were significant correlation between molecular subtypes and nodal status (p=0.03 for luminal B and <0.001 for luminal A, HER 2 positive and triple negative).Conclusion: luminal B and HER2+ve breast cancer subtypes were more likely to be associated with ALNM. Also, tumor size, tumor grade, LVI and ki67 were correlated with lymph node status. Further confirmatory studies are necessary to define factors that predict ALN metastases.

Keywords: Breast cancer, molecular subtypes, axillary lymph nodes metastases.

Introduction
Breast cancer is the most common malignant tumor among women. It has many molecular, pathological and clinical features with different prognostic and therapeutic implications 1. Hormonal receptors (ER, PR) and HER-2/neu have an independent prognostic value. ER expression was demonstrated in 80-90% of breast-cancer cases, while PR expression was demonstrated in 70-80% of cases 2. HER-2/neu over-expressed in about 15-20% of breast cancer cases 3, 4.

Breast cancer is therefore, better represented by its combined receptor expression than by a single receptor status alone 5, 6.Axillary lymph nodal (ALN) metastases are correlated to overall survival and have a strong impact on the staging, prognosis, and treatment of invasive breast cancer 7-9. Breast cancer has at least four main subtypes, Luminal A, Luminal B, Her-2 positive and triple negative breast cancer. Breast cancer molecular subtype (MST) is associated with significant differences in prognosis 10-11.

Triple negative breast cancer has more aggressive clinical behavior, higher metastatic potential and poorer prognosis compared to other subtypes, and characterized by an adverse prognosis particularly in case of limited sensitivity against neoadjuvant chemotherapy 11-13. Despite their aggressive clinical behavior, some studies have shown that lymph node involvement may be less frequent in the triple negative subtype of breast cancer 14, 15.

Many studies have been investigated patients unlikely to benefit from ALN dissection, because the risk of nodal metastasis is low, thus, the use of sentinel lymph node biopsy could reduce the need of ALN dissection at the expense of increasing the risk of missed nodal metastasis. There are controversies about the relation between ALN status and molecular subtypes and role of LN involvement as an intrinsic characteristic. 6, 16The aim of this study is to identify the relation between ALN status and molecular subtype.

Patients and methods:
Three hundred and twenty nine (329) breast cancer patients were included in this study. The study was conducted at Tanta University Hospital, clinical oncology department between January 2011 and January 2015. The clinical and pathological features, including age at diagnosis, menopausal status, tumor site, tumor size, histological type and grade, lymph node status and molecular subtypes were constructed.
Four molecular types were determined according to the current guideline 11.The categorization were made as follows: Luminal A (ER+/PR+, HER2-, Ki67 < 14%); Luminal B (ER+/PR+, HER2+); HER2+ (ER-, PR-, HER2+); TNBC (ER-, PR-, HER2-). IHC 3+ or FISH+ was conceived to be positive HER2 expression.

Statistical analysis: All data were statistically analyzed using the Statistical Package for the Social Sciences, version 21.0 (SPSS Inc., Chicago, IL, USA). The correlation between clinical, pathological features and axillary lymph node status was compared using a Chi-square test. The logistic regression model was used for univariate and multivariate analyses. The P value of less than 0.05 was considered to be statistically significant.

Results
In this study, 329 patients with invasive breast carcinoma were included. The mean age is (50.43) years old. The most histological type is invasive ductal type (86.9%). Luminal A molecular subtype was recorded in 87 patients (26.4%),while luminalB, HER-2+ and TNBC were recorded in 163 patients (49.5%), 35 patients (10.6%) and 44 patients (13.5%) respectively.

One hundred thirty nine patients were pre-menopausal (42.2%), 190 patients were post-menopausal (57.8%). Regarding tumor size , T1 was represented in18.2%, while T2and T3 were recorded in 53.2% and 28.6% respectively. The majority of patients were grade I & II, representing 71.1% while grade III occurred in 28.9%.

Table 1, (Fig. 1) showed that 163 (49.5%) patients had node negative disease while 166 (50.5%) had node positive disease. Nodal stage 2 was the most frequent (106 patients) followed by N1 (34 patients) and26 patients had N3 stage.
Table (1):Distribution of the patients according to N stage (n = 329)
Stage N No. %
N0 163 49.5
N1 34 10.3
N2 106 32.2
N3 26 7.9
N1 + N2 + N3 166 50.5

Distribution of the patients according to N stage (n = 329)(Fig. 1)
Table (2):Relation between N stage and different parameters (n = 329)
Total(n = 329) N Stage p
N0(n = 163) (N1 + N2 +N3) (n = 166) Age ?50 144(43.8%) 50 (34.7%) 94 (65.3%) <0.001*
>50 185(56.2%) 113(61.1%) 72 (38.9%) Sup type Luminal A 87(26.4%) 77(88.5%) 10 (11.5%) <0.001*
Luminal B 163(49.5%) 54(33.1%) 109(66.9%) 0.03*
HER.2+ 35(10.6%) 3(8.6%) 32(91.4%) <0.001*
Triple – 44(13.5%) 29(65.9%) 15(34.1%) <0.001*
Pathology Ductal 286(86.9%) 142(49.7%) 144(50.3%) 0.921
Lobular 43(13.1%) 21(48.8%) 22(51.2%) Menopause Pre 139(42.2%) 52(37.4%) 87 (62.6%) <0.001*
Post 190(57.8%) 111(58.4%) 79 (41.6%) Tsize?2 60 (18.2%) 47(78.3%) 13(21.7%) <0.001*
>2- ?5 175(53.2%) 103(58.9%) 72(41.1%)
>5 94(28.6%) 13(13.8%) 81(86.2%)
Grade G1&2 234(71.1%) 143(61.1%) 91(38.9%) <0.001*
G3 95(28.9%) 20 (21.1%) 75(78.9%) LVI Non 206(62.6%) 142(68.9%) 64(31.1%) <0.001*
Yes 123(37.4%) 21(17.1%) 102(82.9%) Ki67 Low 103(31.3%) 92(89.3%) 11(10.7%) <0.001*
High 226(68.7%) 71(31.4%) 155(68.6%) Table (3) : Univariate analysis logistic regression for factors affecting N stage.

Sig. Exp(B) 95% Confidence Interval for Exp(B)
Lower Bound Upper Bound
age .642 1.176 .593 2.331
menaupaus.268 1.473 .742 2.925
Tumor size .027 .534 .305 .932
LVI <.001 .140 .061 .322
ki67 .067 .155 .018 1.312
pathology .457 .713 .293 1.737
grade .389 .718 .338 1.525
Luminal A .957 .939 .098 8.977
Luminal B .032 .376 .154 .918
HER-2 .038 5.304 1.202 23.406
Triple -ve.157 .326 .069 1.539
Table (4 ): Multivariate analysis logistic regression for factors affecting N stage.

Sig. H.R ratio 95% C.I.for EXP(B)
Lower Upper
suptype(1) .905 1.146 .122 10.814
suptype(2) .030 2.690 1.103 6.561
suptype(3) .015 2.081 1.245 3.479
suptype(4) .144 3.154 .676 14.708
T .033 1.824 1.051 3.167
LVI <.001 6.998 3.050 16.052
Table 2 showed that there are differences in LN positivity by molecular subtypes, (11.5%) of luminal A patients had N positive disease, (66.9%) of luminal B patients, HER2+ (91.4%) and TNBC (34.1%). There were significant correlation between molecular subtypes and nodal status (p=0.03 for luminal B and <0.001 for HER 2 in favors of node positivity and <0.001 for both luminal A and triple negative disease in favors of node negativity).

In the same context, higher tumor grade is significantly associated with ALN metastases, (p<0.001), 34.9% of grade I&II tumors had ALN deposits compared to 78.9% of grade III tumors. There is a significant higher rate of ALN involvement among young age patients (p<0.001), (65.3%) of patients who had ALN metastases were younger than 50 years old compared with (38.9%) in patients older than 50 years old (Table 2).

Correlation between LVI and ALN involvement showed significant relation (p<0.001), (82.9%) of patients with LVI had ALN involvement compared to (31.1%) in patients without LVI. Also there is a significant higher rate of ALN involvement among patients with high ki67 (p<0.001), (68.6%) of patients with high ki67 expression had ALN metastases (Table 2). There was no association between lymph node metastases and tumor pathology.

Table (3) showed univariate logistic regression models. Tumor size, molecular subtypes and LVI showed a significant correlation with the ALN status. In multivariate logistic regression analysis, tumor size (P=0.03), luminal B subtype (P=0.03), HER-2 +ve subtype (P=0.02) and LVI (P<0.001) were independent factors in correlation with the ALN status.Table (4)
Discussion
Breast cancer is a complex, heterogeneous disease at the molecular level17. The immunohistochemistry (IHC) classification of patients has been shown to be correlated well with intrinsic classification using gene expression microarrays: ER/PR+, Her-2- with Luminal A; ER/PR+, Her-2+ with Luminal B; ER-, PR-, Her-2+ (Her-2 positive); and ER-/PR-, Her-2- with Triple negative tumors 1,18,Lymph node (LN) status is one of the most strong factors correlated to overall survival in breast cancer patients, and as such, it has been a major determinant in therapeutic decision making. 20
This study evaluated 329 patients with invasive breast carcinoma, mean age (50.43) years old. The most histological type is invasive ductal carcinoma (86.9%). Luminal A in 87 patients (26.4%), Luminal B in163 (49.5%), HER-2+ in 35 (10.6%), and TNBC in 44 (13.4%) patients.

This study showed that, 163 (49.5%) patients had node negative disease while 166 (50.5%) patients had node positive disease. Nodal stage N2 was the most frequent (106 patients) followed by N1 (34 patients) and 26 patients had N3 stage. The tumor size, grade, menopausal status, LVI and Ki67 expression were significantly correlated the LN positivity. There are differences in LN positivity by molecular subtypes, 10/87 luminal A patients had N positive disease (11.5%), luminal B 109/163 patients (66.9%),HER2+ 32/35 (91.4%) and TNBC 15/44 (34.1%). There were significant correlation between molecular subtypes and nodal status (p=0.03 for luminal B and <0.001 for luminal A, HER 2 + and triple negative).

In our study, there was a lower risk of axillary lymph nodal (ALN) involvement in luminal A and triple negative breast cancer patients and increased frequency of LN metastases in luminal B and HER2+breast cancer patients.
Dent et al 20 showed that patients with Triple negative breast cancer has less lymph node metastases but is more aggressively. It may be due to hematogenous spread or lack of targetable treatment. Chengshuai Si et al 21 study showed that tumor size and tumor subtype show statistical significance with LN involvement. Luminal B type showed significant higher probability of LN involvement, Triple positive and triple negative breast cancer accounts the most and least possibility of LN involvement.

Emi Yoshihara et alHYPERLINK “https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589678/” l “R3″22 study showed that the incidence of ALNM was significantly associated with the presence of LVI (P<0.001), larger tumour size (P < 0.001), higher histologic grade (P < 0.001) and no effect of age. Elsayed M Ali1 et al23study evaluated 258 patients with invasive breast carcinomas, ER and PR expression were demonstrated in 78.7% and 76.4%, respectively and over-expression of HER-2/neu was detected in 13.2% of cases. There was a strong correlation between tumor size and tumor grade with lymph node involvement (p= 0.001 and 0.024, respectively). Triple positive breast cancer is more likely to have axillary lymph node metastases and ER+/PR+/HER-2- (PPN) is the most protected group (p<0.001).

Patani NR et alHYPERLINK “https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589678/” l “R3″24study evaluated 590 patients with mean age 52 years. Positive ALNs were found in 302 patients (51%). Five factors were significantly associated with ALN metastases; younger age, lower mammographic density, higher BI-RADS category, larger tumor size, and presence of lymphovascular invasion. Tufale et alHYPERLINK “https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589678/” l “R3″25evaluated the correlation of various clinicopathological variables with axillary nodal involvement in T1 breast cancer. Tumor size, LVI, histological grade, tumor palpability & ER/PR/Her2 receptor profile were found to be significantly associated with axillary lymph node involvement (ALNI) and also found that age of the patient, family history and histological type of tumor were not significantly correlated with ALNI.

Conclusion:
Analysis of breast cancer subtypes is important, because it provides valuable prognostic and predictive informations. Our results showed that, luminal B ,HER2/neu positive, tumor size and LVI are independent prognostic factors for ALN metastases . Further confirmatory studies are necessary to define factors that predict ALN metastases.

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