A total of 222 breast carcinoma samples were used in the study. The study population were predominantly females with 219/222 (98.6%) being females and 3/222 (1.4%) males. The mean age of patients presenting with breast carcinoma in the cohort was 51.4 ±12.5. A total of 110 (49.5%) involved the right breast, while 107(48.2%) involved the left breast and only 1(0.5) were bilateral. There was no indication of laterality in 4 of the patients. Of the total, 177(79.7%) presented with solitary masses and 41(18.5%) with multiple masses with 4 missing data. Table 1 shows details of clinicopathological features of patients.
Table 1: Clinicopathological features of patients
Parameter
|
Frequency (%)
|
Age (n=220)*
<50
≥50
|
108(49.1%)
112 (50.9%)
|
Grade (n=212)*
I
II
III
|
24(11.3%)
84(39.6%)
104(49.1%)
|
Clinical prognostic staging (n=188)*
IA
IB
IIA
IIB
IIIA
IIIB
IIIC
|
3(1.6%)
9(4.8%)
28(14.9%)
27(14.4%)
25(13.3%)
48(25.5%)
48(25.5%)
|
Nottingham Prog. Index (n=113)*
Good NPI (<3.4)
Moderate NPI (3.41-5.4)
Poor NPI (>/=5.41)
|
3(2.7%)
30(26.5%)
80(70.8%)
|
Lymph Node Stage(n=113)*
1 (Negative)
2 (1-3 LN involved)
3 (> 3 LN involved)
|
26(23.0%)
31(27.4%)
56(49.6%)
|
Vascular Invasion (n=177)*
Yes
No
|
118(66.7%)
59(33.3%)
|
Mitosis(n=164)*
<10
11-20
>20
|
58(35.4%)
63(38.4%)
43(26.2%)
|
Tumour size, cm (n=196)*
≤2
>2- <5
≥5
|
15(7.7%)
63(32.1%)
118 (60.2%)
|
Molecular subtypes (n=212)*
Luminal A
Luminal B
Her2
Triple Negative
|
75 (35.4%)
21 (9.9%)
22 (10.4%)
94 (44.3%)
|
*missing data
Clinicopathological correlations
There was no significant association between age and the clinicopathological features. Apart from clinical prognostic stage (p<0.001), Nottingham Prognostic Index (p<0.001) and Mitosis count (p<0.001) which had significant association with tumour grade, all other clinicopathological parameters did not show any association. Clinical prognostic stage also showed significant association with all clinicopathological parameters (Grade p<0.001; NPI p=0.004; tumour size p<0.001; vascular invasion p<0.001; mitotic count p<0.001) except for weight and age.
Immunohistochemistry analysis
Of the total, 71(32.6%) and 74(33.8%) of patients expressed estrogen and progesterone receptors respectively. 38 (18.3%) patients expressed Her2 receptor. 94 (44.3%) were negative for all 3 receptors hence were triple negative.
Cytoplasmic CD24 expression and its association with clinicopathological features
Eighty-one-point one percent of tumours were cytoplasmic CD24 positive. Tumour grade (p=0.004) and clinical prognostic staging (p=0.027) were the only clinicopathological features which had significant association with CD24 cytoplasmic expression. Age did not have an association with CD24 cytoplasmic expression (OR- 1.9, p=0.064). Grade 2 & 3 tumours had higher CD24 cytoplasmic expression with between 3 to 4-fold increase (OR-3.6, p=0.004). Females predominantly had higher CD24 cytoplasmic expression (OR- 9.5, p=0.028). Although marginally non-significant (p=0.085), it appeared tumours with >10 mitotic features per 10 high power field (/10hpf) had a 2-fold increased CD24 cytoplasmic expression compared with tumours <10/10hpf from the odd ratio. Clinical Prognostic staging (stage III and above) was associated with about 2-fold increased cytoplasmic CD24 expression compared with stage I&II tumours (OR- 2.3, p=0.027). Table 2 summarises the cytoplasmic expression of CD24 and its association with clinicopathological features. Figure 1 shows membranous staining of CD44 and homogenous cytoplasmic staining of CD24
Table 2: CD24 Cytoplasmic expression and its relationship with clinicopathological features
Parameters
|
CD24 Cytoplasmic expression
|
Significance
|
Negative(%) Positive(%)
|
r OR (95%CI) p value
|
Patient’s age
<50
≥50
|
24(61.5)
15(38.4)
|
79(45.1)
96(54.9)
|
0.127 1.9 (0.955-3.957)
|
0.064
|
Grade
1
2/3
|
9(37.5)
26(14.2)
|
15(62.5)
157(85.8)
|
0.199 3.623 (1.410-4.943)
|
0 .004*
|
Tumour size
≤2
>2
|
4(26.7)
30(16.9)
|
11(73.3)
147(83.1)
|
0.068 1.782 (0.531-5.974)
|
0.344
|
|
|
|
|
|
Vascular Invasion
Yes
No
|
19(16.8)
8(13.8)
|
94(83.2)
50(86.2)
|
0 .039 1.263 (0.516-3.090)
|
0.608
|
|
|
|
|
|
LN stage
Negative
Positive
|
5 (19.2)
12(14.3)
|
21(80.8)
72(85.7)
|
0.058 1.429(0.452-4.516)
|
0.542
|
Mitosis
≤10
>10
|
14(24.6)
14(13.7)
|
43(74.5)
88(86.3)
|
0.0136 2.047 (0.896-4.673)
|
0.085
|
NPI
Mod. To Good NPI (<3.4-5.4)
Poor NPI (>/=5.41)
|
2(18.2)
12(15.4)
|
27(81.8)
66(84.6)
|
0.035 1.222 (0.416-3.590)
|
0.715
|
Clinical Prognostic Staging
I&II
III
|
17(25.4)
15(12.6)
|
50(74.6)
104(87.4)
|
0.162 2.357(1.089-5.101)
|
0.027*
|
*statistically significant p value at 95% CI, OR- Odd Ratio, r- Pearson’s correlation coefficient, NPI-Nottingham Prognostic Index
Cytoplasmic CD24 expression and hormone receptor markers
Table 3 indicates the cytoplasmic CD24 expression pattern in relation to hormone receptor status. Only Her2 had a significant association with CD24 cytoplasmic expression. Her2 negative tumours had 5 times higher CD24 expression in comparison with Her2 positive tumours (OR- 4.626, p=0.026). There was no association between the molecular subtypes and CD24 expression (Table 3).
Table 3: Association between CD24 expression, Hormone receptor status and Her2 status
Marker
|
CD24 cytoplasmic expression
|
Significance
|
Negative(%) Positive(%) OR (95%CI)
|
r P Value
|
ER
Positive
Negative
|
13(18.6)
25(17.5)
|
|
57(81.4)
118(82.5)
|
1.076 (0.513-2.295)
|
0.013
|
0.845
|
PR
Positive
Negative
|
13(17.6)
26(18.4)
|
|
61(82.4)
115(81.6)
|
0.943 (0.452-1.965)
|
-0.011
|
0 .875
|
Her2
Positive
Negative
|
2(5.3)
34(20.5)
|
|
36(94.7)
132(79.5)
|
0.216 (0.049-0.941)
|
-0.155
|
0.026*
|
Subtypes
Triple Neg
Others
Molecular subtypes
Luminal A
Luminal B
Her2+
Triple Negative
|
16(17.6)
20(17.1)
16(21.6)
2(9.1)
2(9.1)
16(17.6)
|
|
75(82.4)
97(82.9)
58(78.4)
20(90.9)
20(90.9)
75(82.4)
|
1.035 (0.502-2.132)
1.572(0.758-3.261)
0.447(0.100-2.004)
0.447(0.100-2.004)
1.035(0.502-2.132)
|
0.006
|
0.926
0.222
0.281
0.281
0.926
|
|
*statistically significant p value at 95% CI, OR- Odd Ratio, r- Pearson’s correlation coefficient, ER- Estrogen receptor, PR- Progesterone receptor
Table 4: Association between CD44 and clinicopathological features
Parameters
|
CD44 Cytoplasmic expression
|
Significance
|
Negative(%) Positive(%)
|
OR (95%CI) r p Value
|
Patient’s age
<50
≥50
|
10(47.6)
11(52.4)
|
95(49.0)
99(51.0)
|
0.947 (0.38-2.33) -0.008
|
0.906
|
Grade
1
2&3
|
5(25.0)
15(75.0)
|
18(9.6)
170(90.4)
|
3.148(1.027-9.674) 0.145
|
0 .037*
|
Tumour size
≤2
>2
|
1(5.0)
19(95.0)
|
14(8.2)
157(91.8)
|
0.590 (0.073-4.743) -0.036
|
0.616
|
Vascular Invasion
Yes
No
|
15(78.9)
4(21.1)
|
100(65.4)
53(34.6)
|
1.988 (0.628-6.290) 0.090
|
0.235
|
LN stage
Negative
Positive
|
4(36.4)
7(63.6)
|
22(22.4)
76(77.6)
|
1.974 (0.529-7.367) 0.098
|
0.305
|
Mitosis
≤10
>10
|
10(58.8)
7(41.2)
|
46(31.9)
98(68.1)
|
3.043 (1.089-8.503) 0.173
|
0.028*
|
NPI
Mod. to Good NPI (<5.41)
Poor NPI (>/=5.41)
|
6(54.5)
5(45.5)
|
26(26.3)
73(73.7)
|
3.369 (0.948-11.978) 0.187
|
0.050
|
Triple Negative status
Triple Negative
Others
|
8(8.5)
12(10.3)
|
86(91.5)
105(89.7)
|
0.814(0.318-2.081) -0.030
|
0.667
|
Clinical Prognostic Staging
I &II
III
|
6(9.1)
13(10.7)
|
60(90.9)
108(89.3)
|
0.831(0.300-2.298) -0.026
|
0.721
|
*statistically significant p value at 95% CI, OR- Odd Ratio, r- Pearson’s correlation, NPI-Nottingham Prognostic index
CD24 nuclear staining
CD24 nuclear staining of 14% (30/212) was recorded in this study but did not have an association with any of the clinicopathological features and hormone receptor status.
Association between CD44 expression and clinicopathological features
Tumour grade and mitotic count per high power field were the only clinicopathological parameters significantly associated with CD44 expression. CD44 expression had a 3-fold increase in grades (2&3) tumours (OR-3.148, 95%CI 1.027-9.674, p-0.037). Similarly, a mitotic count > 10/10hpf was found to be associated with higher CD44 cytoplasmic positivity (3-fold increased odds) (OR-3.043 95%CI-1.089-8.503, p=0.028). Table 4 summarises the association between CD44 cytoplasmic expression and clinicopathological features.
Association between CD44 cytoplasmic expression and hormone receptor status
This study revealed no association between CD44 cytoplasmic expression and ER, PR & Her2 status. A summary of CD44 cytoplasmic expression and hormonal status can be found in table 5. Triple negative tumours predominantly expressed CD44 (45%).
Table 5: Association and correlation between cytoplasmic expression of CD44 and hormone receptor status
Marker
|
CD44 cytoplasmic expression
|
Significance
|
Negative (%) Positive(%) OR (95%CI)
|
r p value
|
ER
Positive
Negative
|
9(42.9)
12(57.1)
|
61(31.3)
134(68.7)
|
1.0
1.648 (0.659-4.117)
|
0.073
|
0.282
|
PR
Positive
Negative
|
8(38.1)
13(61.9)
|
64(32.7)
132(67.3)
|
1.0
1.269 (0.501-3.217)
|
0.034
|
0 .615
|
Her2
Positive
Negative
|
4(20.0)
16(80.0)
|
34(18.3)
152(81.7)
|
1.0
1.118 (0.351-3.555)
|
0.013
|
0.850
|
Molecular Subtypes
Luminal A
Luminal B
Her2+
Triple Neg
|
8(40)
3(15)
1(5)
8(40)
|
66(34.6)
18(9.4)
21(11.0)
86(45.0)
|
1.0
0.727 (1.175-3.026)
2.545 (0.301-21.550)
1.303 (0.465-3.654)
|
-0.030
|
0.695
|
*statistically significant p value at 95% CI, OR- Odd Ratio, r- Pearson’s correlation
CD44/CD24 combined phenotypes
Predominantly, CD44+CD24+ was the most occurring combined phenotype (76%) with CD44+CD24- as the second most common phenotype (15%). CD44-CD24-(3%) was however the least occurring phenotype Fig. 2.
Association between CD44/CD24 combined phenotypes and clinicopathological features
Figure 3 shows the various combinations of CD44 and CD24 using same cases as reference.
CD44+CD24-/low
CD44+CD24-/low was found to be significantly associated with tumour grade (OR-3.1, p=0.018), gender (p=0.012), and clinical prognostic staging (OR-2.8, p=0.011). In relation to age, a trend of CD44+CD24-/low appeared to be predominant in the patients <50 years old although marginally non-significant (OR 2.0, p=0.072). Tumours with higher grades (grade 2&3) expressed more CD44+CD24-/low (OR-3.1, p=0.018). Females have 12-fold increased risk of expressing CD44+CD24-/low compared with males (p=0.012). CD44+CD24-/low has almost a 3-fold increased cytoplasmic expression in clinical prognostic stage III compared with lower stages I&II (p=0.011, OR- 2.9)
CD44+CD24+
Significant association of CD44+CD24+ was found with tumour grade (p<0.001 r=-0.246), mitotic count (p=0.017 r=-0.190) and clinical prognostic staging (p=0.040 r=-0.151). Higher grade tumours (2&3) expressed less of CD44+CD24+ phenotype compared to the low grade tumours (OR-0.220, p<0.001). Less CD44+CD24+ expression was seen in the older age group (≥50years) (OR 0.689, p=0.248). The odds of larger tumours (>2cm) expressing CD44+CD24+ phenotype was lesser (OR- 0.617, p=0.398 r=-0.062) likewise the odds of females presenting with this phenotype lesser than males (OR- 0.152 p=0.081). Tumours with <10 mitotic counts/10hpf expressed more CD44+CD24+ phenotypes compared to ≥10 mitotic counts/10hpf (OR-2.46 p= 0.017). Clinical prognostic staging III tumours presented with less CD44+CD24+ phenotypic expression (OR-0.486, p=0.040, r=-0.151).
CD44-CD24-/low
CD44-CD24-/low is significantly associated with mitotic count (p=0.011 r=0.202) and Nottingham Prognostic Index (NPI) (p=0.041 r=0.196). The odds of expression of CD44-CD24-/low is about 10 times higher in tumours with increased mitotic counts (≥10/10hpf). The expression of CD44-CD24-/low corresponded with increasing NPI (OR-7.86 p=0.041)
CD44-CD24+
There is no association between CD44-CD24+ and all the clinicopathological features. Table 6 summarises the CD44CD24 combinatorial phenotypes with clinicopathological features.
Table 6: Association between CD44/CD24 combination phenotypes and clinicopathological features
Parameter
|
|
%CD44+CD24-
|
%ofCD44+CD24+
|
%ofCD44-CD24-
|
%ofCD44-CD24+
|
Age
|
<50(n=101)
≥50 (n=110)
OR
p
|
62.5(n=20)
37.5(n=12)
2.016(0.930-4.372)
0.072
|
45.6(n=73)
54.4(n=87)
0.689(0.366-1.298)
0.248
|
57.1(n=4)
42.9(n=3)
1.471(0.321-6.738)
0.617
|
33.3(n=4)
66.7(n=8)
0.526(0.153-1.806)
0.299
|
Grade
|
1(n=23)
2&3(n=192)
OR
p
|
24.1(n=7)
75.9 (n=22)
3.162(1.170-8.542)
0.018*
|
7.0(n=11)
93.0(n=157)
0.220(0.09-0.539)
0.000*
|
33.3(n=2)
66.7(n=4)
4.214(4.727-24.41)
0.083
|
25.0(n=3)
75.0(n=9)
2.867(0.717-11.466)
0.121
|
Tumour size (cm)
|
≤2(n=15)
>2(n=174)
OR
P
|
14.3(n=4)
85.7(n=24)
2.273(0.669-7.720)
0.178
|
7.0(n=10)
93.0(n=133)
0.617(0.199-1.907)
0.398
|
0.0(n=0)
100.0(n=6)
0.465
|
8.3(n=1)
91.7(n=11)
1.058(0.127-8.806)
0.958
|
Gender
|
Male(n=3)
Female(n=210)
OR
p
|
6.3(n=2)
93.8(n=30)
12.00(1.055-136.49)
0.012*
|
0.6(n=1)
99.4(n=161)
0.152(0.014-1.714)
0.081
|
0.0(n=0)
100.0(n=7)
0.748
|
0.0(n=0)
100(n=12)
0.670
|
Vascular Invasion
|
Present (n=112)
Absent (n=56)
OR
p
|
70.0 (n=14)
30.0 (n=6)
1.190(0.431-3.286)
0.736
|
64.9(n=85)
35.1(n=46)
0.684(0.305-1.537)
0.357
|
71.4(n=5)
28.6(n=2)
1.262(0.237-6.717)
0.785
|
80.0(n=8)
20.0(n=2)
2.077(0.426-10.124)
0.356
|
Lymph Node stage
|
Negative(n=26)
Positive (n=82)
OR
P
|
23.1(n=3)
76.9(n=10)
0.939 (0.238-3.707)
0.929
|
22.4(n=19)
77.6(n=66)
0.658(0.236-1.833)
0.421
|
50.0(n=2)
50.0(n=2)
3.333(0.446-24.93)
0.216
|
33.3(n=2)
66.7(n=4)
1.625
0.585
|
Mitosis
|
≤10(n=55)
>10(n=102)
OR
p
|
40.9(n=9)
59.1(n=13)
1.339 (0.533-3.366)
0.533
|
30.0(n=36)
70.0(n=84)
0.406(0.191-0.863)
0.017*
|
83.3(n=5)
16.7(n=1)
10.100(1.149-88.77)
0.011*
|
55.6(n=5)
44.4(n=4)
2.450(0.630-9.528)
0.184
|
Nottingham Prognostic Index
|
<3.4-5.4 (n=32)
>5.4 (n=77)
OR
P
|
21.4 (n=3)
78.6 (n=11)
0.621(0.161-2.392)
0.485
|
27.1(n=23)
72.9(n=62)
0.618(0.238-1.607)
0.321
|
75.0(n=3)
25.0(n=1)
7.862(0.786-78.673)
0.041*
|
50.0(n=3)
50.0(n=3)
2.552(0.487-13.379)
0.253
|
Triple Negative status
|
Present (n=91)
Absent (n=116)
OR
p value
|
44.8(n=13)
55.2(n=16)
1.042(0.473-2.294)
0.919
|
45.0(n=72)
55.0(n=88)
1.206(0.623-2.334)
0.579
|
42.9(n=3)
57.1(n=4)
0.955(0.208-4.376)
0.952
|
27.3(n=3)
72.7(n=8)
0.460(0.119-1.787)
0.252
|
Clinical prognostic staging
|
I & II (n=66)
III (n=198)
OR
p value
|
22.7(n=15)
77.3(n=51)
2.888(1.239-6.731)
0.011*
|
68.2(n=45)
31.8(n=21)
0.486(0.243-0.973)
0.040*
|
3.0(n=2)
97.0(n=64)
0.898(0.160-5.041)
0.903
|
6.1(n=4)
93.9(n=62)
1.032(0.291-3.665)
0.961
|
*statistically significant p value at 95% CI, OR- Odd Ratio
Association between CD44/CD24 combined phenotypic expression and hormonal status
No significant association is realised between CD44/CD24 combined phenotypes and hormonal status (Table 7).
Table 7: The association between CD44/CD24 combined phenotypic expression and hormonal status
Marker
|
Combinatorial phenotypes expression
|
CD44-/CD24- CD44+/CD24-/low CD44+/CD24+ CD44-/CD24+
|
ER
Positive
Negative
OR
p value
|
3(42.9)
4(57.1)
1.580(0.344-7.261)
0.554
|
10(32.3)
21(67.7)
0.985(0.436-2.224)
0.970
|
50(30.9)
112(69.1)
0.728(0.376-1.411)
0.376
|
6(50.0)
6(50.0)
2.175(0.675-7.008)
0.184
|
|
PR
Positive
Negative
OR
p value
|
3(42.9)
4(57.1)
1.489(0.324-6.840)
0.607
|
10(31.3)
22(68.8)
0.917(0.473-1.775)
0.796
|
54(33.3)
108(66.7)
0.872(0.389-1.957)
0.741
|
5(41.7)
7(58.3)
1.429(0.473-4.670)
0.553
|
|
Her2
Positive
Negative
OR
p value
|
0(0.0)
7(100)
0.195
|
2(6.9)
27(93.1)
0.282(0.064-1.241)
0.076
|
32(20.6)
123(79.4)
1.778(0.694-4.555)
0.226
|
4(36.4)
7(63.6)
2.639(0.731-9.521)
0.126
|
|
Subtypes
Luminal A
Luminal B
Her2+
Triple Neg
|
4(57.1)
0(0.0)
0(0.0)
3(42.9)
|
12(41.4)
2(6.9)
2(6.9)
13(44.8)
|
53(33.1)
16(10.0)
19(11.9)
72(45.0)
|
4(36.4)
3(27.3)
1(9.1)
3(27.3)
|
|
*statistically significant p value at 95% CI, OR- Odd Ratio, ER- Estrogen Receptor, PR- Progesterone receptor