Patient population and clinicopathologic features
The clinicopathological features are summarized in Table 1 and Table 2, and the typical morphological features are shown in Fig. 2. Of the 330 patients, 329 (99.7%) were female. The median age at diagnosis was 48 years (range, 26-79 years). Tumour sizes were most frequent between 0.5 and 5.0 cm (T1–T2), with a median of 2.2 cm. Compared to BM- BCs, BM+ BCs occurred at a younger age (median, 47 vs. 50 years, p=0.031), with larger tumour sizes (median, 2.5 cm vs. 2.0 cm, p=0.024). No significant differences were observed between the groups in tumour stage (pT), lymph node metastasis (pN) or TNM stage. Histologically, of the 210 BCs with available tissue sections, higher histological grade (grade I, 0 vs. 2.3%; grade II, 30.8% vs. 75.8%; grade III, 69.2% vs.22.0%; p=0.008), and necrosis (26.9% vs. 10.6%; p=0.002) were more frequently found in BM+ BCs, while other features, such as LVI, ductal carcinoma in situ (DCIS), calcification, fibrotic focus (FF) and tertiary lymphatic structures (TLS), did not differ between the groups. Regarding clinical management, fewer BM+ BCs received endocrine therapy than BM- BCs (87.2% vs. 74.0%, p=0.002). Other regimens, such as surgical methods, neoadjuvant therapy and adjuvant therapy, did not show significant differences between the groups.
Table 1
Comparison of clinical features of HR+/HER2- breast cancer by BM IHC expression status (n=330).
Characteristics
|
Total (n=330)
|
BM+ (n=150)
|
BM- (n=180)
|
p
|
Age at diagnosis, n (%)
|
|
|
|
|
Median age, years
|
48
|
47
|
50
|
0.031
|
<40
|
49 (14.8)
|
25 (16.7)
|
24 (13.3)
|
0.067
|
40-55
|
194 (58.8)
|
93 (62.0)
|
101 (56.1)
|
|
>55
|
87 (26.4)
|
32 (21.3)
|
55 (30.6)
|
|
pT, n (%)
|
|
|
|
0.061
|
pT1
|
152 (46.1)
|
58 (38.7)
|
94 (52.2)
|
|
pT2
|
156 (47.3)
|
85 (56.7)
|
71 (39.4)
|
|
pT3
|
17 (5.2)
|
5 (3.3)
|
12 (6.7)
|
|
pT4
|
5 (1.5)
|
2 (1.3)
|
3 (1.7)
|
|
pN, n (%)
|
|
|
|
0.412
|
pN0
|
174 (52.7)
|
83 (55.3)
|
91 (50.6)
|
|
pN1
|
94 (28.5)
|
42 (28.0)
|
52 (28.9)
|
|
pN2
|
35 (10.6)
|
10 (6.7)
|
25 (13.9)
|
|
pN3
|
27 (8.2)
|
15 (10.0)
|
12 (6.7)
|
|
TNM staging
|
|
|
|
0.536
|
Ⅰ
|
88 (26.7)
|
34 (22.7)
|
54 (30.0)
|
|
Ⅱa
|
131 (39.7)
|
67 (44.7)
|
64 (35.6)
|
|
Ⅱb
|
47 (14.2)
|
23 (15.3)
|
24 (13.3)
|
|
Ⅲa
|
35 (10.6)
|
9 (6.0)
|
26 (14.4)
|
|
Ⅲb
|
2 (0.6)
|
2 (1.3)
|
0
|
|
Ⅲc
|
27 (8.2)
|
15 (10.0)
|
12 (6.7)
|
|
Surgery, n (%)
|
|
|
|
0.218
|
Breast conservation
|
53 (16.1)
|
20 (13.3)
|
33 (18.3)
|
|
Mastectomy
|
277 (83.9)
|
130 (86.7)
|
147 (81.7)
|
|
Chemotherapy, n (%)
|
|
|
|
0.018
|
Yes
|
265 (80.3)
|
129 (86.0)
|
136 (75.6)
|
|
No
|
65 (19.7)
|
21 (14.0)
|
44 (24.4)
|
|
Endocrine therapy, n (%)
|
|
|
|
0.002
|
Yes
|
268 (81.2)
|
111 (74.0)
|
157 (87.2)
|
|
No
|
62 (18.8)
|
39 (26.0)
|
23 (12.8)
|
|
Radiotherapy, n (%)
|
|
|
|
0.883
|
Yes
|
118 (35.8)
|
53 (35.3)
|
65 (36.1)
|
|
No
|
212 (64.2)
|
97 (64.7)
|
115 (63.9)
|
|
Local relapse n (%)
|
|
|
|
1.000
|
Yes
|
2 (0.6)
|
1 (0.7)
|
1 (0.6)
|
|
No
|
328 (99.4)
|
149 (99.3)
|
179 (99.4)
|
|
Distant Metastasis, n (%)
|
|
|
|
0.047
|
Yes
|
38 (11.5)
|
23 (15.3)
|
15 (8.3)
|
|
No
|
292 (88.5)
|
127 (84.7)
|
165 (91.7)
|
|
Death, n (%)
|
|
|
|
0.055
|
Yes
|
16 (4.8)
|
11 (7.3)
|
5 (2.8)
|
|
No
|
314 (95.2)
|
139 (92.7)
|
175 (97.2)
|
|
Table 2
Association of BM expression with pathological features in HR+/HER2- breast cancer (n=210).
Characteristics
|
Total(n=210)
|
BM+ (n=78)
|
BM- (n=132)
|
p
|
Histological grade, n (%)
|
|
|
|
0.008
|
I
|
3 (1.4)
|
0
|
3 (2.3)
|
|
II
|
124 (59.0)
|
24 (30.8)
|
100 (75.8)
|
|
III
|
83 (39.5)
|
54 (69.2)
|
29 (22.0)
|
|
Ductal carcinoma in situ, n (%)
|
|
|
|
0.050
|
Yes
|
91 (43.3)
|
27 (34.6)
|
64 (48.5)
|
|
No
|
119 (56.7)
|
51 (65.4)
|
68 (51.5)
|
|
lymphovascular invasion (LVI), n (%)
|
|
|
|
0.384
|
Yes
|
73 (34.9)
|
24 (31.2)
|
49 (37.1)
|
|
No
|
136 (65.1)
|
53 (68.9)
|
83 (62.9)
|
|
Uncertain
|
1
|
1
|
-
|
|
Necrosis
|
|
|
|
0.002
|
Yes
|
35 (16.7)
|
21 (26.9)
|
14 (10.6)
|
|
No
|
175 (83.3)
|
57 (73.1)
|
118 (89.4)
|
|
Calcification
|
|
|
|
0.069
|
Yes
|
31 (14.8)
|
7 (9.0)
|
24 (18.2)
|
|
No
|
179 (85.2)
|
71 (91.0)
|
108 (81.8)
|
|
Fibrotic focus (FF)
|
|
|
|
|
Yes
|
37 (17.6)
|
14 (17.9)
|
23 (17.4)
|
0.923
|
No
|
173 (82.4)
|
64 (82.1)
|
109 (82.6)
|
|
Tertiary lymphatic structure (TLS)
|
|
|
|
0.750
|
Yes
|
7 (3.3)
|
3 (3.8)
|
4 (3.0)
|
|
No
|
203 (96.7)
|
75 (96.2)
|
128 (97.0)
|
|
Biomarkers
As shown in Table 3 and Fig. 3, compared to BM- BCs, BM+ BCs were associated with lower expression of ER (0-50% positive, 1.7% vs. 61.3%, p<0.001) and PR (0-50% positive, 33.9% vs. 82.7%, p<0.001). A higher Ki-67 index was also observed in BM+ BCs (Ki-67>50% positive, 40.7% vs. 7.8%, p<0.001). The majority of BM+ BCs was luminal B HER2- subtypes (86.7% vs. 66.1%, p<0.001). BM+ BCs harboured mutated p53 more frequently than BM- BCs (66.7% vs. 40.0%, p<0.001). Among the BM+ BCs, all cases showed EGFR expression while CK5/6 expression was less common (n=78, 52.0% ).
Table 3
Comparison of biomarkers and intrinsic subtypes between BM+ BCs and BM- BCs.
Characteristics, n
|
Total (n=330)
|
BM+ (n=150)
|
BM- (n=180)
|
p
|
ER, n (%)
|
|
|
|
<0.001
|
0-20%+
|
69 (20.9)
|
69 (46.0)
|
0
|
|
21-50%+
|
26 (7.89)
|
23 (15.3)
|
3 (1.7)
|
|
>50%+
|
235 (71.2)
|
58 (38.7)
|
177 (98.3)
|
|
PR, n (%)
|
|
|
|
<0.001
|
0-20%+
|
148 (44.8)
|
105 (70.0)
|
43 (23.9)
|
|
21-50%+
|
37 (11.2)
|
19 (12.7)
|
18 (10.0)
|
|
>50%+
|
145 (43.9)
|
26 (17.3)
|
119 (66.1)
|
|
HER2
|
|
|
|
0.359
|
0
|
209 (63.3)
|
99 (66.0)
|
110 (61.1)
|
|
1+or 2+
|
121 (36.7)
|
51 (34.0)
|
70 (38.9)
|
|
Ki-67, n (%)
|
|
|
|
<0.001
|
0-15%+
|
81 (24.5)
|
20 (13.3)
|
61 (33.9)
|
|
16%-50%
|
174 (52.7)
|
69 (46.0)
|
105 (58.3)
|
|
>50%+
|
75 (22.7)
|
61 (40.7)
|
14 (7.8)
|
|
Intrinsic subtypes, n (%)
|
|
|
|
<0.001
|
Luminal A
|
81 (24.5)
|
20 (13.3)
|
61 (33.9)
|
|
Luminal B HER2-
|
249 (75.5)
|
130 (86.7)
|
119 (66.1)
|
|
BM, n (%)
|
|
|
|
-
|
EGFR positive
|
150 (45.5)
|
150 (100)
|
-
|
|
CK5/6 positive
|
0
|
0
|
-
|
|
Both positive
|
78 (23.6)
|
78 (52)
|
-
|
|
p53 status, n (%)
|
|
|
|
<0.001
|
Mutant-tpye
|
164 (51.6)
|
92 (66.7)
|
72 (40.0)
|
|
Wild-type
|
154 (48.4)
|
46 (33.3)
|
108 (60.0)
|
|
Not known
|
12
|
12
|
0
|
|
Prognosis of BM-positive HR+/HER2- breast cancer
The follow-up time of our cohort ranged from 7.10-72.17 months, with a median of 34.42 months. Survival curves for DFS and OS are shown in Fig. 4. Both DFS and OS were shorter in BM+ BCs (DFS: p=0.034, HR=1.96, 95%CI: 1.04-3.69; OS: p=0.037, HR=2.93, 95%CI: 1.02-8.45). When CK5/6 status was taken into consideration, significantly poorer DFS and OS were also observed (DFS: p=0.033, HR=2.03, 95%CI: 1.06-3.90; OS: p=0.007, HR=3.88, 95%CI: 1.45-1.39) in BM+ BCs. Univariate and multivariate analyses demonstrated that CK5/6 was an independent prognostic factor for both DFS (p=0.004, HR=4.32, 95% CI: 1.66-11.61) and OS (p=0.003, HR=2.82, 95% CI: 1.44-5.55) in HR+/HER2- BCs (Table 4). Meanwhile, clinical stage also played an independent prognostic role in DFS (p<0.001, HR=1.67, 95% CI: 1.27-2.20) and OS (p<0.001, HR=1.86, 95% CI: 1.55-2.24)
Table 4
Univariate analysis and multivariate analysis of HR+/HER2- breast cancer
|
Univariate analysis
|
Multivariate analysis
|
|
p
|
HR
|
95% CI
|
p
|
HR
|
95% CI
|
DFS
|
|
|
|
|
|
|
Clinical stage
|
0.001
|
1.62
|
1.24-2.13
|
<0.001
|
1.67
|
1.27-2.20
|
CK5/6 (pos. vs. neg.)
|
0.007
|
3.88
|
1.45-1.39
|
0.004
|
4.32
|
1.60-11.61
|
EGFR (pos. vs. neg.)
|
0.047
|
2.93
|
1.02-8.45
|
0.580
|
-
|
-
|
ER
|
0.026
|
0.97
|
0.97-1.00
|
0.990
|
-
|
-
|
PR
|
0.010
|
0.98
|
0.96-0.99
|
0.122
|
-
|
-
|
Ki-67
|
0.016
|
1.02
|
1.00-1.04
|
0.585
|
-
|
-
|
OS
|
|
|
|
|
|
|
Clinical stage
|
<0.001
|
1.81
|
1.51-2.15
|
<0.001
|
1.86
|
1.55-2.24
|
CK5/6 (pos. vs. neg.)
|
0.033
|
2.03
|
1.06-3.90
|
0.003
|
2.82
|
1.44-5.55
|
Chemotherapy (yes vs. no)
|
0.030
|
4.85
|
1.17-20.11
|
0.110
|
-
|
-
|
Radiation (yes vs. no)
|
0.001
|
2.86
|
1.51-5.43
|
0.055
|
-
|
-
|
EGFR (pos. vs. neg.)
|
0.038
|
1.96
|
1.04-3.69
|
0.314
|
-
|
-
|
PR
|
0.025
|
0.99
|
0.98-1.00
|
0.175
|
-
|
-
|
Ki-67
|
0.006
|
1.02
|
1.01-1.03
|
0.218
|
-
|
-
|
Among the BM+ patients who received endocrine therapy (n=111, 74.0%), combining adjuvant chemotherapy did not affect their prognosis in terms of either DFS or OS (p=0.457 and 0.670, respectively). On the other hand, it was noteworthy that the OS of BM+ BCs who had been treated with neoadjuvant therapy (n=15, 10.0%) was not improved but rather decreased, compared with those who did not receive the treatment (p=0.037). As the cases were limited, the response to neoadjuvant therapy in BM+ BCs needs further attention.
Immune profiles of BM-positive HR+/HER2- breast cancer
The expression of immunue biomarkers is summarized in Table 5. Of the 210 cases with available tissue blocks for IHC staining, the median sTILs% was 10%. A total of 149 (71.0%) had low sTIL% and 61 (29.0%) had high sTIL%. The sTIL% was notably higher in BM+ BCs than in BM- BCs (50.0% vs.16.7%; p<0.001), with medians of 15% and 5%, respectively. Significantly positive correlations were observed between CD3, CD8, FOXP3, CXCL13, CD68 and BM expression. Examples of immune marker expression and heatmap are displayed in Fig. 5. Among the 61 HR+/HER2- breast cancers, more BM+ BCs showed positive expression of PD-1 (76.9% vs. 50.0%, p=0.031) and PD-L1 (61.5% vs. 32.1%, p=0.010) than BM- BCs.
Table 5
Correlation between BM status and sTILs%, CD3, CD8, FOXP3, CXCL13, PD-1, PD-L1.
N=210
|
Total
(n=210)
|
BM+ (%)
(n=78)
|
BM- (%)
(n=132)
|
Correlation coefficient
|
p
|
TILs
|
|
|
|
0.36
|
<0.001
|
Low
|
149 (71.0)
|
39 (50.0)
|
110 (83.3)
|
|
|
High
|
61 (29.0)
|
39 (50.0)
|
22 (16.7)
|
|
|
CD3
|
|
|
|
0.25
|
<0.001
|
Low
|
132 (62.9)
|
37 (47.4)
|
95 (72.0)
|
|
|
High
|
78 (37.1)
|
41 (52.6)
|
87 (28.0)
|
|
|
CD8
|
|
|
|
0.21
|
0.002
|
Low
|
130 (61.9)
|
38 (48.7)
|
92 (69.7)
|
|
|
High
|
80 (38.1)
|
40 (51.3)
|
40 (30.3)
|
|
|
FOXP3
|
|
|
|
0.26
|
<0.001
|
Low
|
139 (66.2)
|
39 (50.0)
|
100 (75.8)
|
|
|
High
|
71 (33.8)
|
39 (50.0)
|
32 (24.2)
|
|
|
CXCL13
|
|
|
|
0.26
|
0.001
|
Low
|
143 (68.1)
|
41 (52.6)
|
102 (77.3)
|
|
|
High
|
67 (31.9)
|
37 (47.4)
|
30 (22.7)
|
|
|
CD68
|
|
|
|
0.15
|
0.036
|
Low
|
127 (60.5)
|
40 (51.3)
|
87 (65.9)
|
|
|
High
|
83 (39.5)
|
38 (48.7)
|
45 (34.1)
|
|
|
N=61
|
|
|
|
|
|
|
PD-1
|
|
|
|
0.28
|
0.031
|
|
Positive
|
41 (67.2)
|
30 (76.9)
|
11 (50.0)
|
|
|
|
Negative
|
20 (32.8)
|
9 (23.1)
|
11 (50.0)
|
|
|
|
PD-L1
|
|
|
|
0.33
|
0.010
|
|
Positive
|
30 (49.2)
|
24 (61.5)
|
6 (27.3)
|
|
|
|
Negative
|
31 (50.8)
|
15 (38.5)
|
16 (72.7)
|
|
|
|