Download Polymorphisms of glutathione S-transferase P1 (GSTP1) and

Polymorphisms of GSTP1 is associated with differences of chemotherapy
response and toxicity in breast cancer
Bailin ZHANG†, Tong SUN1,†, Baoning ZHANG, Shan ZHENG2, Ning LU2, Binghe
XU3,
Xiang WANG, Guoji CHEN, Dianke YU1, and Dongxin LIN11,*
Center of Breast Diseases and Department of Abdominal Surgery, 1Department of
Etiology and Carcinogenesis, 2Department of Pathology and 3Department of Medical
Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100021, China
*
To whom correspondence should be addressed. Tel: +86 1087788491; Fax: +86
1067722460; Email: [email protected]
†
These authors contributed equally to this work.
1
Abstract
Background Although chemotherapy is one of the most important treatments of breast
cancer, it is limited by significant inter-individuval variations in response and toxicity. The
metabolism of epirubicin (EPI) and cyclophosphamide (CTX) is mainly mediated by cytochrome
P450s (CYPs) and glutathione S-transferases (GSTs). It has been well-known that the activities of
these enzymes are polymorphic in population due to their genetic polymorphisms.
Methods We examined the effects of genetic polymorphisms in CYP3A, GSTP1 and MDR1
genes on treatment response and side-effects of breast cancer patients receiving EPI/CTX
chemotherapy. One hundred and twenty patients with stage II or III invasive breast cancer were
recruited and treated with three to four cycles of EPI 80 mg/m2 and CTX 600 mg/m2 every two
weeks. The AJCC TNM staging system (sixth edition) was used to evaluate the pathological
response of primary tumor and axillary lymph nodes. The genotypes of gene polymorphisms were
determined by using PCR-restriction fragment length polymorphism methods.
Results
Patients carrying GSTP1
105Ile/Val
or
105Ile/Ile
genotype are more likely to have
well response (OR, 0.40; 95% CI, 0.16−0.96; P = 0.024) and light toxicity (OR, 0.35; 95% CI,
0.13−0.78; P = 0.006) than those carrying 105Val/Val genotypes. The response to the treatment was
not correlated with ER, PR and Her2/neu status of tumors. No correlation was found between
toxicity effect and patient’s age, tumor staging, menopause status, and dose intensity of the drugs.
Conclusion
These results suggested that GSTP1 polymorphism was associatiated with the
chemotherapy response or adverse effects of epirubicin and cyclophosphamide regimens.
Key words: Glutathione S-Transferase pi; polymorphisms; Drug Therapy;; Breast Neoplasms;
2
Introduction
Breast cancer is the most frequent cancer of women with an estimation of 1.15 million new
cases globally in 2002[1] and 26% (about 0.19 million) of all new cancer cases among women in
the United States in 2009[2]. Although the prognosis of breast cancer is good as the advance of
systematic therapy[3, 4], significant heterogeneity in the response and toxicity of chemotherapeutic
agents is also observed[5]. Resistance to chemotherapy and toxicity of specific agents are largely
determined by multifaceted enzymatic systems that are cytotoxic targets or members of the
metabolic pathway of the administered drug. In spite of many clinical characters (e.g., age, organ
function, tumour biology, concurrent medications), genetic differences in drug transport,
metabolism and drug targets also contribute to the difference of treatment outcomes[6].
Most patients with breast cancer were treated with chemotherapy especially those with
locally advanced disease. Anthracycline and cyclophosphamide (CTX) based chemotherapy
regimen is commonly used as recommended by NCCN clinical practice guidelines of breast cancer.
The toxicity profile of this regimen is characterized by myelosuppression, cardiotoxicity and
urotoxicity. Epirubicin can produce a cytotoxic effect through intercalation with DNA, eventually
inducing DNA cleavage by topoisomerase II. Doxorubicin is a substrate of P-glycoprotein,
encoded
by
the
multidrug
resistance
(MDR-1)
gene.
Activation
of
CTX
to
4-hydroxy-cyclophosphamide (4-OH-CPA) is catalyzed mainly by the hepatic cytochrome P450
(CYP) isozymes. 4-OH-CPA interconverts rapidly with its tautomer, aldophosphamide and it is
likely that both of these metabolites passively diffuse out of hepatic cells, circulate, and then
passively enter other cells[7].
Glutathione S-transferases (GSTs), a superfamily of dimeric phase II metabolic enzymes,
play an important role in the cellular defense system. GST enzymes catalyze the conjugation of
toxic and carcinogenic electrophilic molecules with glutathione and thereby protect cellular
macromolecules from damage. The subclass GSTP1 is widely expressed in normal human
epithelial tissues. The genetic changes of GSTP1 may alter the function of the enzyme. Deleted or
mutated GSTP1 may be associated with less detoxification of CTX, resulting in more available
drug compared to the wild-type enzyme. Cytochromes in the P4503A family are estimated to
participate in the metabolism of 40 to 60% of all clinically administered drugs. Neoadjuvant
(preoperative) chemotherapy provides an opportunity to directly assess tumor response and
3
toxicity to therapy without interference of other treatments.
On the basis of these preclinical and clinical data, we hypothesized that genetic variants in
the major drug-metabolizing enzyme involved in EPI and CTX predict interindividual variability
treatment response. To test this hypothesis, we examined the association between therapy response,
toxicity and the genetic polymorphisms in CYP3A, GSTP1 and MDR1 from 120 patients received
neoadjuvant chemotherapy of EPI and CTX regimen. We found that GSTP1 polymorphism was
associatiated with chemotherapy response or adverse effects of epirubicin and cyclophosphamide
regimens.
4
METHODS
Patients and treatment regimen
From June 2005 to March 2007, one hundred and twenty patients with stage II or III invasive
breast cancer were recruited and treated with three to four cycles of EPI 80 mg/m2 and CTX 600
mg/m2 every two weeks. Core needle biopsy of primary breast tumor was performed and
anticoagulated peripheral blood was obtained from each patient before the starting of treatment.
The patients were aged from 29 to 70 years and had normal haematopoietic, cardiac, pulmonary,
renal and hepatic functions. All patients signed an informed consent prior to entering the study.
This study was approved by the Institutional Review Board of the Chinese Academy of Medical
Sciences Cancer Hospital and Institute.
Response assessment and toxicity evaluate criteria
Clinical response of tumor was evaluated according to the Response Evaluation Criteria in
Solid Tumors (RECST)[8]. The complete response was defined as disappearance of tumor for at
least four weeks; partial response—at least a 30% decrease of the longest diameter of tumor for
more than 4 weeks; progressive disease—at least a 20% increase of the longest diameter of tumor;
stable disease—neither sufficient shrinkage to qualify for partial response nor sufficient increase
to qualify for progressive disease. The sixth edition of American Joint Committee on Cancer
(AJCC) tumor– node– metastasis (TNM) staging system[9] was used to evaluate both clinical stage
before chemotherapy and pathological response of primary tumor and axillary lymph nodes after
the treatment ( Table 1 ). Patients with stage 0 to II and stage III were defined as well and poor
responses respectively. Chemotherapy related toxic reaction was evaluated according to the
Common Terminology Criteria for Adverse Events (CTCAE v3.0)[10]. Patients with grade I, II and
Grade III, IV were defined as general and severe toxicity respectively.
Genotyping
Genomic DNA was extracted from blood samples of all patients. All assays were performed
using a polymerase chain reaction based restriction fragment length polymorphism (PCR–RFLP)
technique twice. Such genomic DNAs (50 ng each) were amplified by PCR, and each PCR
5
product was digested with the appropriate enzyme (Table 2) according to the manufacturer’s
protocol. After restriction enzyme analysis PCR fragments were visualized in a 2–3% agarose gel.
The assays for six polymorphisms in CYP3As, GSTP1 and MDR1 genes were described in Table
2.
Statistical analysis
Pearson’s chi-square test was used to examine the differences in tumor response or
chemotherapy toxicity among different genotypes of the genetic polymorphisms, and the
associations were estimated using odds ratios (ORs) and their 95% confidence intervals (CIs).
Differences in age distribution, tumor stage, menopausal statues and axillary nodes metastasity
among patients with different toxicity after chemotherapy were also analyzed with Pearson’s
chi-square test. The t-test was used to evaluate difference of average dose intensity of CTX and
EPI between patients with different response and toxicity. The P value <0.05 was used as the
criterion of statistical significance. All statistical tests were two-sided and performed with
computer programs from Statistical Analysis System (SAS Institute, Cary, NC, USA).
6
RESULTS
A total of 120 patients with a median age of 49 years (range: 29 – 70 years) were evaluated in
this study. The clinicopathological features of patients are summarized in Table 3 and 4. All
patients were ethnic Han women. The clinical response rate of this group was 75.8%, with seven
patients (5.8%) of complete response and eighty-four patients (70.0%) of partial response.
Twenty-nine (24.2%) patients had stable disease. No patient had disease progression during
treatment. In this group, 70 patients (58.3%) had well response and 50 patients (41.7%) received
poor response. The pathological complete response (stage 0) rate was 5.8%. All patients received
the same regimen during chemotherapy. Estrogen receptor, progesterone receptor status and
expression of the HER2 protein were measured by immunohistochemical analysis. Only complete
membrane staining of invasive tumor cells was considered in the results. As the result of HER2
was not confirmed with fluorescent in situ hybridization analysis, only tumor cells scored three
plus on the immunohistochemical analysis were considered to be positive for overexpression of
the HER2 protein.
There was no statistical significance of age, clinical tumor down-staging, menopausal status
and average dose intensity of CTX and EPI between patients with severe (Grade III or IV) and
average (Grade I or II) toxicity or different response. The modified radical operation was
performed 111 patients (92.5%). The other 9 patients (7.5%) received breast conserving treatment.
Two patients failed to be genotyped in more than one polymorphic site because of PCR
amplification problems with their DNA samples. Records of toxicity were not available in five
patients treated in outpatient department. Patients carrying GSTP1 105Ile/Val or 105Ile/Ile genotype
are more likely to have well response (OR, 0.40; 95% CI, 0.16−0.96; P = 0.024) and light toxicity
(OR, 0.35; 95% CI, 0.13−0.78; P = 0.006) than those carrying
105Val/Val
genotypes. In patients
with poor response and well response, 38 (76.0%) and 38 (55.9%) carried GSTP1
genotype respectively. In patients with well response, 29 (42.6%) were
(1.5%) was
105Ilel/Ile
carriers. The
105Ile/
Val and
105Ile/Ile
105Ile/Val
105Val/Val
carrier and 1
genotype carriers in poor response
group were 10 (20.0%) and 2 (4.0%) respectively (Table 5). The
105Val/Val
carrier numbers in
severe and common toxicity groups were 41 (77.4%) and 32 (52.5%) respectively. 12 cases
7
(22.6%) with 105Ile/Val genotype suffered from the serve toxicity and no one in this group carried
the
105
Ile/Ile genotype. Among patients with average toxicity, 26 (42.6%) and 3 subjects (4.9%)
carried
105
Ile/Val and
105
Ile/Ile genotype respectively. No significant difference was found among
other polymorphic sites.
8
DISCUSSION
Although chemotherapy improves disease-free and overall survival from breast cancer
patients[11], there are also great challenges that to identify patients who do benefit from
chemotherapy and reduce the use of chemotherapy in those who do not derive benefit. In locally
advanced breast cancer, the use of preoperative systemic chemotherapy has been shown to induce
tumor response and facilitate local control through subsequent surgery and radiation therapy.
Preopearative chemotherapy is established as the standard of care for patients with locally
advanced breast cancer[12, 13]. Breast cancer comprises a spectrum of related but different cancer
subtypes, which have different causal genetic changes, may follow different clinical courses, and
require different treatments tailored to the phenotype[14, 15].
We initially hypothesized that functional polymorphisms in CYP3As, GSTP1 and MDR1 gene
would lead to distinct phenotypes of drug metabolism that would predict outcome to
chemotherapy in breast cancer patients. We need to find out some other predictive factors to help
the decision of individual treatment. In this study, we found that genetic variability in GSTP1 was
significantly associated with treatment response and chemotherapy toxicity to CTX and EPI
regimen. Patients with GSTP1
105Val/Val
genotype were more likely to have poor response and
severe toxicity. The contribution of genotypes on outcome was of statistical significance. These
findings suggest that genetic variation in drug metabolism may play an important role in
chemotherapy efficacy in breast cancer.
As the mechanism of EPI metabolism is rarely reported, CTX metabolism always serves as a
paradigm for the role of drug-metabolizing enzymes to predict treatment response[16]. GSTs and
multiple hepatic cytochrome P450s play important roles in activation of CTX. GSTP1 is the most
abundant GST found in many normal and malignant tissues[17]. Many chemotherapeutic agents,
including CTX and anthracyclines are substrates for GSTP1. Polymorphic of single-nucleotide
substitutions in the coding sequence of GSTP1 (1578 A>G) give rise to Ile105Val amino acid
substitutions which lies within the substrate-binding site of GSTP1[17,
18].
The GSTP1
105Val
variant is associated with a lower thermal stability and altered catalytic activity to a variety of
substrates compared with GSTP1 105Ile[19]. Patients with homozygous isoleucine (Ile/Ile) have the
highest level of GSTP1 activity. The activity is somewhat reduced in heterozygotes (Ile/Val) and
further diminished for those with two copies of valine (Val/Val). Our results showed that patients
9
with GSTP1
105
Val/Val genotype were more likely to have poor response and severe toxicity.
Besides the breast cancer[20], it is further supported by the trend of GSTP1
105
Ile genotypes with
higher activity contribute to an well prognosis of multiple myeloma[21] and ovarian cancers[22]
treated with at least one agent in our study regimens. In spite of the poor therapeutic response,
diminished enzyme activity of GSTP1 also induces a severe toxicity. This finding is consistent
with the hypothesis that patients with the GSTP1
105
Val variant enzyme have a reduced ability to
detoxify chemotherapeutic agents, which result in the lower clearance and reduced efficacy[23].
It is reported interpatient variability in exposure to CTX correlated to treatment outcome. But
result of a resent study did not support blood drug level of anticancer agents correlating with
genotype and phenotype of drug metabolizing enzymes[24]. To our best knowledge, there is no
report on the relationship
between polymorphisms of
drug metabolizing enzymes,
pharmacokinetics of anticancer agents and observed variability in clinical response and toxicity of
breast cancer treatment. Besides above mentioned finding, Angela reported that combined
genotypes at CYP3A4, CYP3A5, GSTM1, and GSTT1 influenced the probability of treatment
failure after high-dose adjuvant chemotherapy for node-positive breast cancer. Patients who
carried low-drug genotype group had a 4.9-fold poorer disease-free survival (P = 0.021) and a
four-fold poorer overall survival (P = 0.031) compared with individuals who carried high-drug
genotype group[16].
In summary, our study provides information about the role of GSTP1 polymorphisms in
outcome after neoadjuvant therapy of breast cancer. Confirmation of these findings and supportive
mechanistic data will ultimately allow the potential for drug metabolism enzyme genotyping to be
realized in the clinic to individualize and optimize breast cancer therapy.
Acknowledgements
This work was supported by Beijing Municipal Science and Technology grant D0905001040131
10
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12
Table 1. Revised 2003 AJCC TNM staging system for breast cancer as applied to evaluate
pathologic extent of disease after neoadjuvant chemotherapy
Tumor and nodal categories
T0N0 (including residual DCIS)
T1N0
T0-1N1; T2N0
T2N1; T3N0
T0-3N2; T3N1
Any T4
Any N3
Stage
0
I
IIA
IIB
IIIA
IIIB
IIIC
Nodal metastases (N) reflect both the number and the presence of involved axillary lymph nodes.
DCIS: ductal carcinoma in situ.
13
Table 2. PCR primers and restriction enzymes for genotyping SNPs
Gene name
CYP3A4*1G
CYP3A5*3
CYP3A7*2
GST P1
MDR1 Ex12
C1236T
MDR1 Ex26
C3435T
SNPs
Primers
rs2242480
C>T
rs776746
A>G
rs2740565
A>T
rs1695
Ile105Val
rs1128503
C>T
rs34748655
C>T
F: 5’-AGGGATTTGAGGGCTTCACT-3’
R: 5’-CAGAGCCAGCACGTTTTACA-3’
F: 5’- CATGACTTAGTAGACAGATGA-3’
R: 5’- GGTCCAAACAGGGAAGAAATA -3’
F: 5’-TCTATAAAGTCACAATCCCTGAGACCTGATTCATG -3’
R: 5’- GCCAAAGAGTGAGCTCAAAAA -3’
F: 5’- TGAATGACGGCGTGGAGGAC -3’
R: 5’- GGGGTGAGGGCACAAGAAGC -3’
5’- TATCCTGTGTCTGTGAATTGCC -3’
5’- CCTGACTCACCACACCAATG -3’
5’- TGTTTTCAGCTGCTTGATGG -3’
5’- AAGGCATGTATGTTGGCCTC -3’
Restriction
enzyme
Rsa I
Ssp I
BspH I
Mae II
Hae III
Mo I
14
Table 3. The clinicopathological features among patients with different treatment response
Well response
n = 70
Poor response
n = 50
N, (%)
N, (%)
P
Age ( years)
≤ 45
46−55
> 55
Median
Tumor stage at diagnosis
II
III
Menopausal status at diagnosis
Premenopausal
Postmenopausal
Estrogen receptor status**
Positive
Negative
Progesterone receptor status**
Positive
Negative
Her2 expression status**
Overexpression
Non overexpression
Average dose intensity (Mean ± SD†)
CTX (mg / m2)
EPI (mg / m 2)
Toxicity ‡
Grade III or IV
Grade I or II
27 (38.6)
30 (42.8)
13(18.6)
49
19 (38.0 )
21(42.0)
10 (20.0)
49
48 (68.6)
22 (31.4)
26(52.0)
24(48.0)
0.066*
47 (67.1)
23 (32.9)
32 (64.0)
18 (36.0)
0.720*
40 (58.8)
28 (41.2)
35 (70.0)
15 (30.0)
0.213
46 (67.6)
22 (32.4)
41 (82.0)
9 (18.0)
0.080
19 (27.9)
49 (72.1)
13 (26.0)
37 (73.0)
0.815
595 ± 33
77.2 ± 4.4
597 ± 34
77.9 ± 2.5
0.773#
0.285#
28 (43.1)
37 (56.9)
26 (52.0)
24 (48.0)
0.342*
0.981*
* Two-sided chi-square test.
# Two-sided t test.
† Standard Deviation
‡ Records of toxicity were not available in five patients.
** Data of two patients in well response group was not available.
15
Table 4. The clinicopathological features among patients with different toxicity
Severe toxicity
n = 54
Average toxicity
n = 61
N, (%)
N, (%)
P
Age
≤ 45
46−55
> 55
Median
Clinical tumor down-staging
Yes
No
Axillary nodes metastasity
Positive
Negetive
Average dose intensity (Mean ± SD†)
CTX (mg / m2)
EPI (mg / m2)
20 (37.0)
24 (44.5)
10 (18.5)
47
23 (37.7)
23 (37.7)
15 (24.6)
49
39 (72.2)
15 (27.8)
45 (73.8)
16 (26.2)
0.852*
38 (70.4)
16 (29.6)
42 (68.9)
19 (31.1)
0.860*
602 ± 33
77.7 ± 3.2
592 ± 31
76.3 ± 7.3
0.107#
0.177#
0.668*
* Two-sided chi-square test.
# Two-sided t test.
† Standard Deviation
16
Table 5. Allelic and genotypic frequencies of CYPs, MDR1 and GST among patients with well or poor response, difference toxicity and the association
with therapeutic effect of chemotherapy
Genotype
GSTP1
(rs1695)
Val/Val
Ile/Val
Ile/Ile
Ile/Val+ Ile/Ile
Ile allele frequence
CYP3A5*3
(rs776746)
AA
GA
GG
GA + GG
A allele frequence
CYP 3A4*1G
(rs2242480)
CC
CT
TT
CT + TT
T allele frequence
Well response
n (%)
Poor response
n (%)
38(55.9)
29(42.6)
1(1.5)
30(44.1)
0.228
38(76.0)
10(20.0)
2(4.0)
12(24.0)
0.140
Reference
0.34(0.13−0.87)
2.00(0.13-58.34)
0.40(0.16−0.96)
32(47.1)
31(45.5)
5(7.4)
36(52.9)
0.699
27(54.0)
20(40.0)
3(6.0)
23(46.0)
0.740
Reference
0.76(0.33-1.75)
0.71(0.12-3.89)
0.76(0.34-1.68)
38(54.3)
28(40.0)
4(5.7)
32(45.7)
0.257
29(58.0)
20(40.0)
1(2.0)
21(42.0)
0.220
Reference
0.94(0.41−2.12)
0.33(0.01−3.42)
0.86(0.39−1.91)
*
OR (95% CI)
P#
Severe toxicity Average toxicity
(Grade III or IV) (Grade I or II) OR† (95% CI)
n (%)
n (%)
P#
0.012
0.571
0.024
41(77.4)
12(22.6)
0
12(22.6)
0.113
32(52.5)
26(42.6)
3(4.9)
29(47.5)
0.262
Reference
0.36(0.14-0.89)
0.00(0.09-1.89)
0.35(0.13-0.78)
0.014
0.056
0.006
0.489
0.722
0.456
28(52.8)
25(47.2)
0(0.0)
25(47.2)
0.764
29(47.5)
25(41.0)
7(11.5)
32(52.5)
0.680
Reference
0.97(0.42-0.2.21)
0.928
1.24(0.55-2.76)
0.573
0.863
0.393
0.686
28(51.9)
25(46.3)
1(1.8)
26(48.1)
0.250
36(59.0)
21(34.4)
4(6.6)
25(41.0)
0.238
Reference
1.52(0.67-3.52)
0.32(0.01-3.37)
1.34(0.60-2.99)
0.273
0.300
0.440
17
CYP3A7*2
(rs2740565)
AA
AT
TT
AT+TT
T allele frequence
MDR1Ex12C1236T
(rs1128503)
TT
CT
CC
CT+CC
C allele frequence
MDR1Ex26C3435T
(rs34748655)
CC
CT
TT
CT+TT
C allele frequence
33(47.8)
30(43.5)
6(8.7)
36(52.2)
0.304
28(54.9)
21(41.2)
2(3.9)
23(45.1)
0.245
Reference
0.82(0.36-1.87)
0.39(0.05-2.43)
0.75(0.34-1.66)
27(38.6)
35(50.0)
8(11.4)
43(61.4)
0.364
14(28.0)
29(58.0)
7(14.0)
36(72.0)
0.430
Reference
1.60(0.66-3.90)
1.69(0.43-6.61)
1.61(0.69-3.81)
23(33.3)
33(47.8)
13(18.9)
46(66.7)
0.428
22(44.0)
20(40.0)
8(16.0)
28(56.0)
0.360
Reference
0.63(0.26-1.53)
0.64(0.10-1.74)
0.64(0.28-1.44)
0.616
0.451
0.443
25(46.3)
28(51.9)
1(1.8)
29(53.7)
0.278
35(57.4)
19(31.1)
7(11.5)
26(42.6)
0.270
Reference
2.06(0.88-4.84)
0.20(0.01-1.82)
1.56(0.70-3.49)
0..066
0.111
0.235
0.256
0.391
0.229
21(38.9)
26(48.1)
7(13.0)
33(61.1)
0.370
18(29.5)
34(55.7)
9(14.8)
43(70.5)
0.426
Reference
0.66(0.27-1.59)
0.67(0.18-2.49)
0.66(0.28-1.53)
0.306
0.496
0.289
0.266
0.412
0.236
17(31.5)
25(46.3)
12(22.2)
37(68.5)
0.454
28(46.7)
23(38.3)
9(15.0)
32(53.3)
0.342
Reference
1.79(0.72-4.46)
2.20(0.68-7.21)
1.90(0.83-4.41)
0.166
0.140
0.098
* ORs of poor response.
# Two-sided chi-square test.
† ORs of severe toxicity.
18