Citations

Abstract The selective estrogen receptor modulators (SERM) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high-risk women by 50%, but this U.S. Food and Drug Administration-approved prevention therapy is not often used. We attempted to identify genetic factors that contribute to variation in SERM breast cancer prevention, using DNA from the NSABP P-1 and P-2 breast cancer prevention trials. An initial discovery genome-wide association study identified common single-nucleotide polymorphisms (SNP) in or near the ZNF423 and CTSO genes that were associated with breast cancer risk during SERM therapy. We then showed that both ZNF423 and CTSO participated in the estrogen-dependent induction of BRCA1 expression, in both cases with SNP-dependent variation in induction. ZNF423 appeared to be an estrogen-inducible BRCA1 transcription factor. The OR for differences in breast cancer risk during SERM therapy for subjects homozygous for both protective or both risk alleles for ZNF423 and CTSO was 5.71. Significance: This study identified novel, functionally polymorphic genes involved in the estrogen-dependent regulation of BRCA1 expression, as well as a novel mechanism for genetic variation in SERM therapeutic effect. These observations, and definition of their underlying mechanisms, represent steps toward pharmacogenomically individualized SERM breast cancer prevention. Cancer Discov; 3(7); 812–25. ©2013 AACR. See related commentary by Machiela and Chanock, p. 728 This article is highlighted in the In This Issue feature, p. 705

Abstract Introduction: The NSABP P-1 and P-2 breast cancer prevention trials have demonstrated that the SERMs tamoxifen and raloxifene can reduce the risk of breast cancer in high risk women. Methods: We performed a nested matched case-control GWAS utilizing DNA from participants enrolled in P-1 and P-2. 592 participants developed invasive breast cancer or ductal carcinoma in situ, and were matched with 1171 controls who did not. Genome-wide genotyping with the Illumina 610-Quad chip and CYP2D6 genotyping for pertinent alleles were performed. Functional genomic studies involved the use of cultured cells, with siRNA knockdown, followed by qRT-PCR and Western blot analyses. Results: Eleven SNPs with p-values < 3E-05 were identified. Twenty one additional SNPs with p < 4E-05 were identified by imputation around the genotyped SNPs on chromosomes (Chrs) 4, 8, 9, 13, 16 and 22, which were then genotyped with the Invader platform. Initial functional genomic studies focused on 6 SNPs on Chr16 (p-values 1.81-9.55E-06), all of which were in ZNF423, a gene encoding a putative zinc-finger protein. The Chr16 minor variant SNP sequences had less risk than the common variant (OR=0.7). Our functional studies showed that incubation of Hs578T cells stably transfected with estrogen receptor (ER) α with 0.1 nM estradiol (E2) induced expression of ZNF423, BRCA1/2 for WT but not variant SNP sequences. There was also a striking difference between E2-induced ZNF423, BRCA1/2 expression for variant and WT SNPs in lymphoblastoid cell lines (LCLs) stably transfected with ERα, with only WT showing induction. ChIP assay showed that ZNF423 could bind to the 5’-flanking region of BRCA1, and reporter gene assays showed that ZNF423 could regulate BRCA1/2 transcription. In LCLs stably transfected with ERα and genotyped for ZNF423 SNPs, estrogen-dependent expression of all 3 genes occurred only in the presence of the WT, but not variant ZNF423 SNP sequences. Blockade of ERα with 4-OH tamoxifen prevented induction of expression in cells with WT sequences, but resulted in gene dose-dependent increases in BRCA1/2 expression in cells with variant SNP sequences-suggested that the minor variant has a “protective” effect during clinical SERM therapy and verified by ChIP assays. Conclusions: The SNPs in ZNF423 identified during GWAS were associated with differential E2-dependent induction of ZNF423, BRCA1 and BRCA2-implying that ZNF423 is “upstream” for the estrogen-dependent induction of BRCA1/2 expression. However, LCLs with variant SNP sequences showed greatly enhanced expression of ZNF423, BRCA1/2 during 4-OH tamoxifen exposure, while cells with WT sequences did not. These results may reveal a novel mechanism for SERM-dependent regulation of BRCA1/2 expression and may have implications for patient selection for SERM therapy. Funded by U10-CA37377, U10-CA69974 and U14-GM61388. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2011-4727

Background— Preclinical diastolic dysfunction (PDD) has been broadly defined as subjects with left ventricular diastolic dysfunction, without the diagnosis of congestive heart failure (HF) and with normal systolic function. Our objective was to determine the risk factors associated with the progression from PDD (stage B) HF to symptomatic (stage C) HF. Methods and Results— Using the resources of the Rochester Epidemiology Project, all residents of Olmsted County, MN, who underwent echocardiography between January 1, 2004, and December 31, 2005, and had grade 2–4 diastolic dysfunction and ejection fraction ≥50% were identified. Patients with a diagnosis of HF before or within 30 days of the echocardiogram were excluded. Patients were also excluded if they had a diagnosis of atrial fibrillation or severe mitral or aortic valve regurgitation at the time of the echocardiogram. A total of 388 patients met the inclusion criteria. The mean age of the cohort was 67±12 years, with a female (57%) predominance. Prevalence of renal insufficiency (estimated glomerular filtration rate <60 mL/min per 1.73 m 2 ) was 34%. The 3-year cumulative probabilities of development of (stage C) HF, development of atrial fibrillation, cardiac hospitalization, and mortality were 11.6%, 14.5%, 17.7%, and 10.1% respectively. In multivariable Cox proportional hazard regression analysis, we determined that age, renal dysfunction, and right ventricular systolic pressure were independently associated with the development of HF. Conclusions— This population-based study demonstrated that in PDD (stage B) HF, there was a moderate degree of progression to symptomatic (stage C) HF over 3 years, and renal dysfunction was associated with this progression independent of age, sex, hypertension, coronary disease, and ejection fraction.

Abstract Background: Controversy exists regarding the association between CYP2D6 enzyme activity and tamoxifen effectiveness in the adjuvant treatment of invasive breast cancer; however, this association in the primary prevention of breast cancer is unknown. Methods: We conducted a nested case–control study in the context of the NSABP P1 and P2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, and metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both noninvasive and invasive) while on 5 years of selective estrogen receptor modulators therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was conducted for alleles associated with absent (*3, *4, *5, and *6), reduced (*10, *17, and *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded. Results: A total of 591 cases were matched to 1,126 controls and DNA was genotyped in more than 97%. In patients treated with tamoxifen, there was no association of CYP2D6 genotype [OR (extensive/poor metabolizer): 0.90; 95% CI: 0.46–1.74, P = 0.74), use of a potent CYP2D6 inhibitor (OR 0.92; 95% CI: 0.575–1.486), or CYP2D6 metabolizer status (OR 1.03; 95% CI: 0.669–1.607) with breast cancer occurrence. Likewise, there was no association between any CYP2D6 metabolism parameter with breast cancer events in raloxifene-treated patients. Conclusion: In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy. Clin Cancer Res; 17(21); 6944–51. ©2011 AACR.

Abstract Background: NSABP P1 and P2 are the largest prevention trials of tamoxifen and raloxifene in women at high risk of developing breast cancer. Both agents have been shown to decrease, but not eliminate breast cancer risk in these women. The goal of our study was to identify single nucleotide polymorphisms (SNPs) associated with development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Methods: We performed a nested matched case-control genome-wide association study (GWAS) in subjects entered on the P2, which included only postmenopausal women, and subjects at least 50 years old entered on the P1 phase III prevention trials who were treated with tamoxifen or raloxifene. Cases were defined as subjects who developed invasive breast cancer or ductal carcinoma in situ and each was matched to two controls based on trial (P1, P2), age at trial entry, Gail risk score, history of lobular carcinoma in situ, and time on study. The study was restricted to the 94% of subjects self-identified as Caucasian. Genotyping was performed with the Illumina Human610-Quad chip. Eigenstrat analyses were performed to control for population stratification. SNPs were imputed within 200 Kb on either side of regions containing SNPs with p ≤ 3E-05. CYP2D6 genotyping was performed on all subjects. Results: The GWAS included 592 cases and 1171 controls. 547,356 SNPs were used in the analyses after removing 14,019 for call rates less than 98%, 30,560 for minor allele frequencies &lt;1%, and 301 with Hardy-Weinberg equilibrium p-value &lt;1E-06. Eigenvectors did not alter the results when used as covariates. Eleven genotyped SNPs with p-values ranging from 2.12E-06 to 9.98E-06 (conditional logistic regression with eigenvector adjustment) were identified; and 22 SNPs with p &lt; 4E-05 were identified by imputation around these SNPs on chromosomes (Chr) 4, 8, 9, 13, 16 and 22, which were then genotyped with the Invader platform. Following imputation and genotyping, initial functional genomic studies have focused on 8 SNPs on Chr 16 (p-values 1.81E-06 to 9.55E-06, with the lowest p-value for an imputed and fine mapped SNP), all of which were in ZNF423, a gene known to encode a protein that associates with SMAD family members. In lymphoblastoid cell lines genotyped for the 2 top SNPs in ZNF423 (rs8060157 and rs11076499) and stably transfected with estrogen receptor-α, the variant and wild type SNPs displayed striking differences in estradiol-induced expression of ZNF423, SMAD3, BRCA1, and BRCA2. Conclusions: This GWAS revealed SNPs associated with the development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Genome-wide significance was not achieved, but SNPs in ZNF423 identified on Chr 16 were associated with differential expression of ZNF423, SMAD3, BRCA1, and BRCA2 in response to estradiol in vitro, providing direction for future research. (Supported in part by NIH grants U01GM61388, U01GM63173, P50CA116201, U10CA77202, U10CA37377, U10CA69974, U24CA114732, and the Biobank Japan Project funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-02.

Abstract Abstract #76 Background: QOL and MH outcomes were integrated into the NSABP B-30 trial as secondary outcomes to the efficacy analyses which are being presented separately. Explicit secondary aims of the NSABP B-30 study were 1) to compare toxicities among the regimens, 2) to compare QOL, and 3) to examine differences in amenorrhea and its relationship to symptoms, QOL, and efficacy. Here we examine the secondary aims of the study as a companion to the efficacy results that are presented separately.&#x2028; Materials and Methods: 5351 pts with cT1-3, N0-1, M0 were enrolled from 3/1/99 to 3/31/2004. 2170 were enrolled on the QOL study, and 2449 were enrolled on the MH study and were randomized to one of three treatment groups: Group 1 [doxorubicin (A) 60 mg/m2 and C 600 mg/m2 q 3 weeks (wks) x 4 followed by docetaxel (T) 100 mg/m2 q 3 wks x 4; Group 2 [A 50 mg/m2 and T 75 mg/m2 q 3 wks x 4]; Group 3 [A 50 mg/m2 T 75 mg/m2 and cyclophosphamide (C) 500 mg/m2 q 3 wks x 4]. All patients with ER-positive tumors received hormonal therapy after completing chemotherapy. Preliminary results from Group 1 have been reported previously (Swain, et al. Breast Cancer Res Treat, 2008).&#x2028; Results: The protocol specifies that 800 deaths are required for the definitive analysis of treatment, QOL, and MH outcomes, which are expected to occur by fall 2008. For this final report, results from a comparison of the three arms will be analyzed and presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 76.

Abstract Abstract #75 Background: The primary aims of this study were to determine 1) if a concurrent regimen of therapy containing cyclophosphamide compared to a sequential regimen containing cyclophosphamide would prolong disease-free survival (DFS) and overall survival (OS) and 2) if a regimen without cyclophosphamide was at least as efficacious as the two regimens containing cyclophosphamide. The secondary aims were to compare toxicities among the three regimens and to compare differences in amenorrhea in premenopausal women in each arm and its relationship to outcome.&#x2028; Materials and Methods: 5351 pts with cT1-3, N0-1, M0 were enrolled from 3/1/99 to 3/31/2004. 1783 pts were randomized to Group 1 [doxorubicin (A) 60 mg/m2 and C 600 mg/m2 q 3 weeks (wks) x 4 followed by docetaxel (T) 100 mg/m2 q 3 wks x 4], 1784 pts to Group 2 [A 50 mg/m2 and T 75 mg/m2 q 3 wks x 4], and 1784 to Group 3 [A 50 mg/m2 T 75 mg/m2 and cyclophosphamide (C) 500 mg/m2 q 3 wks x 4]. All patients with ER-positive tumors received hormonal therapy after completing chemotherapy. The protocol specifies that 800 deaths are required for the definitive analysis. This is expected to occur by Fall 2008.&#x2028; Results: Mean time on study is currently 79 months. At entry, 46% of pts were &lt; 49 yrs; 54% were &gt; 50 yrs. 82% were white, 10% black, 4% Hispanic. 66% had 1-3 positive nodes, 26% had 4-9, and 8% had 10+. 75% of pts were ER pos. 49% had lumpectomy, 51% mastectomy, and 24% local/regional radiotherapy. Mean tumor size was 2.5 cm. The presentation will include the first results of DFS, OS, and relevant toxicities from the trial. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 75.

Abstract Abstract #6140 Background:&#x2028; Ixabepilone (ixa), the first in a new class of antineoplastic agents, showed clinical benefit in combination with capecitabine (C) in 2 large clinical trials in metastatic breast cancer (MBC) pts whose tumors were resistant to (study 046, JCO, 2007) or pretreated with anthracycline (A) and taxanes (T) (study 048). In 048, ixa + C, compared to C alone, demonstrated significant increases in PFS (HR 0.79 [0.69-0.90]) and ORR (43% vs 29%). A trend toward increased OS was seen both in 048 (HR 0.90 [0.78-1.03]) and in 046 (HR 0.90 [0.77-1.05]), which did not reach statistical significance. Similar to taxanes, dose-limiting peripheral neuropathy (PN) is a side effect associated with ixa treatment. Here we present the incidence rates, management of ixa-induced PN from 3 registrational studies in pts with MBC and an analysis of resolution time. Also, data on potential risk factors for PN are presented.&#x2028; Methods:&#x2028; Incidence of PN is summarized from a 2000 pt program from 3 multi-center clinical trials. In this program, almost 1100 pts with MBC received ixa either as a monotherapy (40 mg/m2 IV over 3h Q3w, N=126) in a phase II study, or in combination with C (1000 mg/m2 PO BID x14d Q3w) in 2 large phase III studies (N = 369 and N = 595). PN was evaluated every 4 weeks after completion of treatment until toxicity was resolved. Resolution of PN was defined as the time from onset of worst grade to baseline or grade 1. A Cox regression analysis was also performed on a dataset of 945 pts with different tumor types across multiple studies, receiving ixa as a monotherapy or in combination with C, to identify the potential risk factors for grade 3/4 PN.&#x2028; Results:&#x2028; Incidence rates of grade 3/4 PN and resolution time observed in 1 phase III study (046) and the phase II (081) have been published before (081: JCO, 25; 3407). Here we present the data from the final confirmatory phase III study (048).&#x2028; &#x2028; PN was effectively managed with dose reduction. After dose reduction, symptoms improved or did not worsen in the majority of pts, and a median of 2-3 additional treatment cycles were delivered. Pts with diabetes were at a higher risk of grade 3/4 PN (HR: 1.67 [1.00-2.78], p-value = 0.049).&#x2028; Conclusions:&#x2028; Incidence of ixa-associated PN was similar to that observed in trials of other T based regiments. Ixa-induced PN has a predictable median time to resolution of 5-6 weeks over 3 large studies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6140.

Abstract A recent genome-wide association study found that genetic variants on chromosomes 3, 6, 7, 10, 11, 19 and X were associated with prostate cancer risk. We evaluated the most significant single-nucleotide polymorphisms (SNP) in these loci using a worldwide consortium of 13 groups (PRACTICAL). Blood DNA from 7,370 prostate cancer cases and 5,742 male controls was analyzed by genotyping assays. Odds ratios (OR) associated with each genotype were estimated using unconditional logistic regression. Six of the seven SNPs showed clear evidence of association with prostate cancer (P = 0.0007-P = 10−17). For each of these six SNPs, the estimated per-allele OR was similar to those previously reported and ranged from 1.12 to 1.29. One SNP on 3p12 (rs2660753) showed a weaker association than previously reported [per-allele OR, 1.08 (95% confidence interval, 1.00-1.16; P = 0.06) versus 1.18 (95% confidence interval, 1.06-1.31)]. The combined risks associated with each pair of SNPs were consistent with a multiplicative risk model. Under this model, and in combination with previously reported SNPs on 8q and 17q, these loci explain 16% of the familial risk of the disease, and men in the top 10% of the risk distribution have a 2.1-fold increased risk relative to general population rates. This study provides strong confirmation of these susceptibility loci in multiple populations and shows that they make an important contribution to prostate cancer risk prediction. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2052–61)