Loss of constitutive ABCB1 expression in breast cancer associated with worse prognosis

Figures & data

Figure 1 Flowchart of the study cohort.

Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; IHC, immunohistochemistry.

Figure 2 Immunohistochemical evaluation of ABCB1 in human tissues.

Notes: The right column indicates 100× magnification, and the left column indicates 400× magnification. (A and B) Immunostaining of ABCB1 in the liver; (C and D) immunostaining pattern control of a specific marker for biliary canaliculi (CD10) in the liver; (E and F) negative controls of ABCB1 in tonsils; (G and H) controls of ABCB1 in the kidney – proximal (strong reaction) and distal (weak reaction) tubules are positive controls, and glomeruli (black arrows) are negative controls; (I and J) immunostaining of ABCB1 in a breast cancer sample, showing regions with positive (brown) and negative reactions (circumvented by a red line), as they were seen in the slide (I) or side-by-side for better visualization (J). All the sections were counterstained with hematoxylin (blue staining).
Abbreviation: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1.

Table 1 Description of individual features in breast cancer patients

Clinical features	n	%	Histopathological features	n	%
Age at diagnosis			Histological type
24–34 years	15	2.1	Ductal invasive	597	83.9
35–44 years	82	11.5	Lobular invasive	40	5.6
45–54 years	181	25.4	Ductal in situ	45	6.3
55–64 years	208	29.2	Lobular in situ	3	0.4
≥65 years	226	31.7	Others	27	3.8
Color			Missing	1
White	320	45.7	Tumor size (pT)
Brown	268	38.3	pTis (in situ)	48	6.7
Black	88	12.6	pT1 (≤2 cm)	321	45.1
Others	24	3.4	pT2 (2–5 cm)	309	43.4
Missing	12		pT3 (>5 cm)	21	2.9
Menopausal status			Missing	13
Premenopausal	179	25.6	Lymph node status (pN)
Postmenopausal	521	74.4	pN0 (none)	421	59.6
Missing	12		pN1 (≤3 positive nodes)	177	25.1
Smoking status			pN2 (4–9 positive nodes)	66	9.3
Nonsmoker	464	66.2	pN3 (≥10 positive nodes)	42	5.9
Former smoker	173	24.7	Missing	6
Current smoker	64	9.1	Tumor grade (G)
Missing	11		G1	83	12.7
Alcohol consumption			G2	261	39.9
Never	384	56.0	G3	310	47.4
Rare (<3 days/week)	212	30.9	Missing	58
Frequent (>3 days/week)	90	13.1	Stage grouping
Missing	26		0	46	6.6
Comorbidities			I	227	32.5
None	91	12.8	II	312	44.7
Hypertension	377	53.3	III	113	16.2
Obesity	159	28.7	Missing	14
Diabetes mellitus	104	14.9	ER status
BMI (Kg/m^2)			Negative	111	16.2
<18.4	6	1.0	Positive	575	83.8
18.5–24.9	164	28.4	Missing	26
25–29.9	248	43.0	PR status
>30.0	159	27.6	Negative	197	28.8
Missing	135		Positive	488	71.2
ABCB1 genotypes			Missing	27
rs1128503 (C1236T)			HER-2 status
CC	222	39.7	Negative	515	81.1
CT	269	48.1	Positive	103	16.2
TT	68	12.2	Undetermined	17	2.7
			Missing	77
rs1045642 (C3435T)			Tumor subtype
CC	227	39.3	Luminal A	395	63.2
CT	257	44.5	Luminal B	130	20.8
TT	94	16.3	HER-2-like	38	6.1
			Triple-negative	62	9.9
			Missing	87

Notes: n=712. Data are expressed as the number and percentage of patients in each category.

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; BMI, body mass index; ER, estrogen receptor; HER-2, human epidermal growth factor receptor 2; PR, progesterone receptor.

Figure 3 Photomicrographs of ABCB1 immunostaining in human mammary tissue samples showing different immunoreaction intensities.

Notes: (A–H) Breast cancer samples: (A and B) negative; (C and D) weak; (E and F) moderate; (G and H) strong immunostaining; (I and J) nonmalignant mammary tissues with strong immunostaining.
Abbreviation: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1.

Figure 4 Distribution of ABCB1 immunoreaction scores in human breast tumors.

Notes: (A) immunoreaction score (IRS); (B) continuous scale (CS).
Abbreviation: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1.

Figure 5 Distribution of ABCB1 mRNA levels in human breast tumors.

Notes:ABCB1 mRNA levels are shown according to the ABCB1 IRS (A); ABCB1 CS (B); rs1128503 genotypes (C); and rs1045642 genotypes (D). *p<0.5; **p<0.01. Both in Mann-Whitney test.
Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; CS, continuous scale; IRS, immunoreaction score; RQ, relative quantification.

Table 2 Significant associations between individual features and ABCB1 expression in breast tumors

Individual features	IRS status		CS
	OR	95% CI	Median	95% CI	pM-W
Hypertension
No	1		200	160–250	<0.001
Yes	0.51	0.32–0.82	180	103–239
Tumor size (pT)
pT1	1		200	135–269	0.003
pT2 + pT3	0.59	0.37–0.95	185	155–250
Lymph node status (pN)
pN0 + pN1	1		190	123–294	0.001
pN2 + pN3	3.64	1.44–9.19	202.5	120–240
Stage grouping
I + II	1		190	120–240	<0.001
III	3.15	1.34–7.43	210	170–270
ER status
Positive	1		195	145–250	<0.001
Negative	0.38	0.23–0.65	160	70–220
PR status
Positive	1		200	150–250	<0.001
Negative	0.47	0.30–0.76	177.5	90–220
Tumor subtype
Luminal A	1		195	150–250
Luminal B	0.86	0.46–1.62	190	143–245	<0.001a
HER-2-like	0.82	0.27–2.47	195	140–238
Triple-negative	0.21	0.11–0.39	125	20–193

Notes: n=656. Data are expressed as the OR, based on the IRS status, or as the median value of the CS, for each category, with the respective 95% CIs. Statistically significant differences are presented in bold characters.

a p-value of Kruskal–Wallis test.

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; CI, confidence interval; CS, continuous scale; ER, estrogen receptor; IRS, immunoreaction score; HER-2, human epidermal growth factor receptor 2; OR, odds ratio; pM-W, p-value for the Mann–Whitney test; PR, progesterone receptor.

Table 3 Multivariate model of ABCB1 positivity according to clinical and histopathological features of breast tumors

Individual features	Adjusted OR	Adjusted 95% CI	Wald coefficient	pH-L
Triple-negative
Positive	0.24	0.13–0.45	19.6
Hypertension
Yes	0.42	0.24–0.73	9.7	0.298
Lymph node status (pN)
pN0 + pN1	0.27	0.10–0.71	7.1	0.552
Tumor size (pT)
pT2 + pT3	0.55	0.32–0.93	4.9	0.139

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; CI, confidence interval; OR, odds ratio; pH-L, p-value for the Hosmer–Lemeshow test.

Table 4 Influence of hypertension severity and treatment on ABCB1 positivity in breast tumors

Individual features	IRS status		CS
	OR	95% CI	Median	95% CI	pM-W
Hypertension treatment
Normotensive	1		200	160–250	<0.001a
Monotherapy	0.76	0.40–1.43	180	125–243
Combined therapy	0.58	0.33–1.03	188	110–236
Resistant hypertensionb	0.17	0.08–0.35	160	45–225
Diuretics (n)
No (168)	1		180	103–240	0.890
Yes (180)	0.57	0.312–1.02	185	103–230
ACE inhibitors (n)
No (190)	1		185	108–245	0.432
Yes (154)	1.09	0.61–1.92	180	100–226
Beta-blockers (n)
No (227)	1		185	110–240	0.234
Yes (117)	0.68	0.38–1.22	180	85–230
ATR1 antagonists (n)
No (257)	1		180	110–230	0.967
Yes (87)	0.87	0.46–1.64	180	90–250
Calcium channel blockers (n)
No (278)	1		180	100–236	0.657
Yes (66)	1.36	0.63–2.92	187.5	130–240

Notes: n=344. Data are expressed as the OR, based on the IRS status, or as the median value of the CS, for each category, with the respective 95% CIs. Statistically significant differences are presented in bold characters. The number of patients (n) per group are given in parentheses.

a p-value of Kruskal–Wallis test;

b Classified according to Daughert.

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; ACE, angiotensin converting enzyme; ATR1, angiotensin II receptor 1; CI, confidence interval; CS, continuous scale; IRS, immunoreaction score; OR, odds ratio; pM-W, p-value for the Mann–Whitney test.

Figure 6 Two-year disease-free survival curves of patients with breast cancer based on either ABCB1 protein levels (IRS) or dichotomized mRNA expression.

Notes: (A) IRS on all tumors; (B) IRS on triple-negative tumors; (C) mRNA levels on all tumors. p log-rank and HR with respective 95% CIs are provided for the comparison between positive IRS (green on A and B) and negative IRS (blue on A and B) or between mRNA levels above the median (high in green on C) or below the median (low in blue on C).
Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; CI, confidence interval; CS, continuous scale; HR, hazard ratio; IRS, immunoreaction score.

Table 5 Influence of ABCB1 mRNA status (low) on the disease-free survival of breast cancer cohorts available at ProgGeneV2.0

Study ID	n	Follow-up (years)	DFS	OS	Adjusting factor	Reference
NKI	295	16	p<0.0001; HR =1.5 (1.2–1.8)	p<0.0002; HR =1.6 (1.2–1.9)	ER	^Citation57
GSE11121	200	16	p<0.01; HR =3.8 (1.4–10.0)	N/A	None	^Citation58
GSE4922_U133A	249	11	p=0.003; HR =2.8 (1.4–5.6)	N/A	ER	^Citation59
GSE22219	215	1	p=0.003; HR =3.8 (1.6–9.1)	N/A	ER and grade	^Citation60
GSE17705	298	16	p=0.001; HR =9.1 (2.3–33.3)	N/A	None	^Citation61
GSE2034	246	13	p=0.6; HR =1.1 (0.7–2.0)	N/A	ER	^Citation62
GSE3494_U133A	236	11	N/A	p=0.006; HR =3.6 (1.4–8.3)	ER and PR	^Citation63
GSE21653	247	13	N/A	p=0.004; HR =1.7 (1.2–2.4)	ER	^Citation64
TCGA-BRCA	594	13	N/A	p=0.2; HR =1.12 (0.7–1.1)	ER and PR	^Citation65

Notes: Data were obtained from survival plots generated on ProgGeneV2.0 for cohorts with at least 200 patients.

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; DFS, disease-fee survival (relapse-free or metastases-free, as available for each study); ER: estrogen receptor; HR, hazard ratio; N/A, not available; OS: overall survival; p, p-value for log-rank analysis; PR, progesterone receptor.

Table 6 Influence of ABCB1 mRNA status (low) on the 5-year disease-free survival and overall survival of breast cancer cohorts available at the GEO database

Subset of breast cancer patients	DFS	OS
All
n	3,951	1,402
p log-rank	1e–16	0.00025
Hazard ratio	1.89 (1.67–2.13)	1.64 (1.25–2.13)
ER positive
n	2,061	548
p log-rank	0.0018	0.095
Hazard ratio	1.35 (1.12–1.64)	1.47 (0.93–2.33)
ER negative
n	801	251
p log-rank	0.00071	0.027
Hazard ratio	1.54 (1.19–1.92)	1.82 (1.06–3.13)
Basal
n	618	241
p log-rank	2.4e–07	0.0081
Hazard ratio	2.00 (1.54–2.63)	2.17 (1.2–3.85)

Notes: Data were obtained from survival plots generated on KM plotter by using the filter for breast cancer. Cutoff values for low and high ABCB1 mRNA levels were set to the median.

Abbreviations: ABCB1, adenosine 5′-triphosphate–binding cassette subfamily B member 1; DFS, disease-fee survival; ER, estrogen receptor; GEO, the Gene Expression Omnibus; OS, overall survival.

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