Yoon-Sim Yap

Whole-exome sequencing of breast cancer, malignant peripheral nerve sheath tumor and neurofibroma from a patient with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by the development of multiple neurofibromas, cafe‐au‐lait spots, and Lisch nodules. Individuals with NF1 are at increased risk of developing various tumors, such as malignant peripheral nerve sheath tumor (MPNST), pheochromocytoma, leukemia, glioma, rhabdomyosarcoma, and breast cancer. Here, we describe the exome sequencing of breast cancer, MPNST, and neurofibroma from a patient with NF1. We identified a germline mutation in the NF1 gene which resulted in conversion of leucine to proline at amino acid position 847. In addition, we showed independent somatic NF1 mutations in all the three tumors (frameshift insertion in breast cancer (p.A985fs), missense mutation in MPNST (p.G23R), and inframe deletion in dermal neurofibroma (p.L1876del‐Inf)), indicating that a second hit in NF1 resulting in the loss of function could be important for tumor formation. Each tumor had a distinct genomic profile with mutually exclusive mutations in different genes. Copy number analysis revealed multiple copy number alterations in the breast cancer and the MPNST, but not the benign neurofibroma. Germline loss of chromosome 6q22.33, which harbors two potential tumor suppressor genes, PTPRK and LAMA2, was also identified; this may increase tumor predisposition further. In the background of NF1 syndrome, although second‐hit NF1 mutation is critical in tumorigenesis, different additional mutations are required to drive the formation of different tumors.

Predictors of Hand-Foot Syndrome and Pyridoxine for Prevention of Capecitabine–Induced Hand-Foot Syndrome: A Randomized Clinical Trial

Importance Hand-foot syndrome (HFS) is a common adverse effect of capecitabine treatment. Objective To compare the incidence and time to onset of grade 2 or greater HFS in patients receiving pyridoxine vs placebo and to identify biomarkers predictive of HFS. Design, Setting, and Participants This single-center, randomized double-blind, placebo-controlled phase 3 trial conducted at National Cancer Centre Singapore assessed whether oral pyridoxine could prevent the onset of grade 2 or higher HFS in 210 patients scheduled to receive single-agent capecitabine chemotherapy for breast, colorectal, and other cancers. Interventions Patients were randomized to receive concurrent pyridoxine (200 mg) or placebo daily for a maximum of 8 cycles of capecitabine, with stratification by sex and use in adjuvant or neoadjuvant vs palliative setting. Patients were withdrawn from the study on development of grade 2 or higher HFS or cessation of capecitabine. Main Outcomes and Measures Primary end point was the incidence of grade 2 or higher HFS in patients receiving pyridoxine. Secondary end points included the time to onset (days) of grade 2 or higher HFS and identification of biomarkers predictive of HFS, including baseline folate and vitamin B12 levels, as well as genetic polymorphisms with genome-wide arrays. Results In this cohort of 210 patients (median [range] age, 58 [26-82] years; 162 women) grade 2 or higher HFS occurred in 33 patients (31.4%) in the pyridoxine arm vs 39 patients (37.1%) in the placebo arm (P = .38). The median time to onset of grade 2 or higher HFS was not reached in both arms. In univariate analysis, the starting dose of capecitabine (odds ratio [OR], 1.99; 95% CI, 1.32-3.00; P = .001), serum folate levels (OR, 1.27; 95% CI, 1.10-1.47; P = .001), and red blood cell folate levels (OR, 1.25; 95% CI, 1.08-1.44; P = .003) were associated with increased risk of grade 2 or higher HFS. In multivariate analyses, serum folate (OR, 1.30; 95% CI, 1.12-1.52; P < .001) and red blood cell folate (OR, 1.28; 95% CI, 1.10-1.49; P = .001) were the only significant predictors of grade 2 or higher HFS. Grade 2 or higher HFS was associated with 300 DNA variants at genome-wide significance (P < 5 × 10−8), including a novel DPYD variant (rs75267292; P = 1.57 × 10−10), and variants in the MACF1 (rs183324967, P = 4.80 × 10−11; rs148221738, P = 5.73 × 10−10) and SPRY2 (rs117876855, P < 1.01 × 10−8; rs139544515, P = 1.30 × 10−8) genes involved in wound healing. Conclusions and Relevance Pyridoxine did not significantly prevent or delay the onset of grade 2 or higher HFS. Serum and red blood cell folate levels are independent predictors of HFS. Trial Registration clinicaltrials.gov Identifier: NCT00486213

Abstract CT045: Ribociclib + letrozole for first-line treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC): efficacy by baseline tumor markers

Background: Cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes promote cell proliferation through phosphorylation of retinoblastoma protein (Rb). In breast cancer, cyclin D-CDK4/6 activity can be increased through cyclin D gene (CCND1) amplification or loss of the CDK4/6 negative regulator p16. Here we present efficacy data from the Phase III MONALEESA-2 study of ribociclib (CDK4/6 inhibitor) + letrozole vs. placebo + letrozole for first-line treatment of HR+, HER2- ABC, assessed in baseline tumors by protein levels of Rb, p16, the cell proliferation marker Ki67, and by gene expression levels of CDKN2A (p16) and CCND1. Methods: Postmenopausal women with HR+, HER2- ABC with no prior systemic therapy for advanced disease were randomized 1:1 to receive ribociclib or placebo (600 mg/day 3-weeks-on/1-week-off) + letrozole (2.5 mg/day continuous). The primary endpoint was investigator-assessed progression-free survival (PFS). Provision of a representative baseline tumor biopsy or archival tissue at screening was mandatory if available. Baseline tumor tissue was evaluated for protein biomarkers (immunohistochemistry) and gene expression (NanoString nCounter® Human Cancer Reference panel). Results: Of 668 patients randomized, 479 were evaluable for total Rb, and 416 (87%) displayed high levels (H-score ≥100). p16 protein levels were evaluable in 405 patients; 165 (41%) had low (H-score 14% of cells in 247 (53%) patients. The median messenger RNA expression level was used as the cut-off to define patients with low or high baseline CDKN2A and CCND1 gene expression. An improved PFS was observed by the addition of ribociclib to letrozole in all the above patient subgroups, with hazard ratios ranging from 0.40 (high p16 by H-score; 95% confidence interval [CI] 0.16-1.0; p=0.06) to 0.64 (≤14% Ki67-positive cells; 95% CI 0.39-1.0; p=0.07). Patients with less or greater than 14% Ki67-positive cells, lower or higher p16 levels, Rb levels, or CDKN2A or CCND1 gene expression benefitted from the addition of ribociclib to letrozole to a similar extent. Conclusions: A consistent benefit from ribociclib + letrozole vs. placebo + letrozole was observed irrespective of baseline Rb, p16, and Ki67 levels or CDKN2A and CCND1 gene expression levels. Hormone receptor positivity remains the only established biomarker of response to CDK4/6 inhibitors. Citation Format: Fabrice Andre, Salomon M. Stemmer, Mario Campone, Katarina Petrakova, Shani Paluch-Shimon, Yoon-Sim Yap, Norbert Marschner, Arlene Chan, Cristian Villanueva, Lowell L. Hart, Carlos L. Arteaga, Gabe S. Sonke, Eva-Maria Grischke, Emilio Alba, Arnd Nusch, Denise A. Yardley, Erik Jakobsen, Sibel Blau, Sara M. Tolaney, Faye Su, Wei He, Caroline Germa, Gabriel N. Hortobagyi. Ribociclib + letrozole for first-line treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC): efficacy by baseline tumor markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT045. doi:10.1158/1538-7445.AM2017-CT045

Abstract 2923: Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients

Background Label-free methods for isolating circulating tumor cells (CTCs) are attractive because they provide an opportunity to analyze a larger set of CTCs that may otherwise be missed due to variable or no expression of protein (label) markers. Understanding genetic and functional heterogeneity in CTCs allows us to gain insight into the mechanisms underscoring metastasis, drug resistance, and tumor aggressiveness. Currently, a simple workflow for isolation and molecular characterization of single CTCs by mRNA sequencing is lacking. In order to address this challenge, we developed a label-free workflow to isolate CTCs from breast cancer patients for full-length mRNA sequencing analysis by integrating the ClearCell® FX System with the Polaris™ system. The ClearCell FX system processes blood samples from cancer patients and enriches for CTCs in a label-free antibody-independent manner. The low level of nonspecifically isolated white blood cells from ClearCell FX is further depleted on the Polaris system by negative enrichment of viable CTCs. This unique integration of systems will enable researchers to perturb single CTCs in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. Method and Results CTCs from 7.5 mL of peripheral blood sample from breast cancer patients were enriched using ClearCell FX. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Single CTCs were selected on Polaris (Fluidigm) system, lysed and reverse-transcribed, and cDNA were preamplified on the Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated using the Nextera® kit and sequenced on Illumina® MiSeq™ and NextSeq™ systems. We successfully processed blood samples from four patients. Sequenced data showed high-quality metrics, with read depth of up to 2.5 million reads (MiSeq) or 60 million reads (NextSeq), with a low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchical clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient. We will provide insights into full-length mRNA transcriptome of single CTCs from triple negative breast cancer patient. Conclusion We present the feasibility of integrating two microfluidics platforms to capture single CTCs for transcriptome and functional study. Our data suggests that the heterogeneity of tumor sample and characterization of metastatic processes can be elucidated from single-cell mRNA sequencing of CTCs. Citation Format: Naveen Ramalingam, Yi Fang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Jovina Tan, Chong Tracy Lu, Ninez Delos Angeles, Chad Sanada, Cassandra Greene, Kyle Hukari, Andrew Wu, Yoon-Sim Yap, Jay West, Ali Asgar Bhagat. Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2017-2923

Validation of the AJCC 8th prognostic system for breast cancer in an Asian healthcare setting

AIMS We aim to validate the AJCC 8th edition prognostic staging system for breast cancer in an Asian setting. METHODS Clinico-pathologic information and cancer-specific survival (CSS) outcomes of 6287 stage I to III patients with invasive breast cancer who underwent upfront surgery at SingHealth institutions in Singapore from 2006 to 2014 were analyzed. Survival distributions for the different staging systems were estimated by the Kaplan-Meier method and compared using the log-rank tests. Multivariable Cox proportional hazards models were used, with Akaike Information Criterion (AIC) and Harrells Concordance Index (C-index) to compare both staging systems. Among patients with positive hormone-receptor status, 84.8% received endocrine therapy. Among the cohort, 60.3% of received chemotherapy; 82.1% of node positive patients received chemotherapy and 86.0% of HER2-enriched patients in whom chemotherapy was also indicated received adjuvant HER2-targeted therapy. Ninety-seven percent of patients received anthracyclines and/or taxanes containing chemotherapy regime. RESULTS The median follow up was 64 months. 2921 patients (46.5%) were discordant between the anatomic and prognostic systems of which 363 (5.8%) were upstaged and 2558 (40.7%) were down-staged. For all patients, stages in both the prognostic and anatomic systems were discriminating for 5-year CSS. Controlling for age, ethnicity and receipt of chemotherapy, the prognostic staging system model (AIC = 7538.87, C = 0.79) presented slightly better explanation and concordance of survival times than the anatomic staging system model (AIC = 7607.31, C = 0.77). CONCLUSION The prognostic staging system was better than the anatomic staging system in predicting outcomes but the anatomic system remains relevant due to its ease of use.

Abstract B22: Inducing cell state transitions in triple-negative breast cancer (TNBC)

Triple-negative breast cancer (TNBC), as immunohistochemically defined by its estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and human epidermal growth factor receptor-2 (HER2)-negative status, is an important subtype due to its biologically aggressive behavior and limited treatment options available. TNBC is associated with an overall poorer prognosis, with higher risk of disease recurrence/progression and shorter duration of treatment response, i.e., treatment resistance. Treatment resistance may be largely attributed to cancer stem cells (CSCs), which are intrinsically treatment resistant and continually self-renew, proliferate, and differentiate into different phenotypes. Activation of the cell biologic program, epithelial-mesenchymal transition (EMT), has been demonstrated to promote the dedifferentiation of heterogeneous subpopulations of cancer cells towards CSC phenotypes. We hypothesized that induction of the mesenchymal-epithelial transition (MET) program might disrupt CSC function, drive differentiation, and render greater susceptibility to conventional chemotherapy. In this study, we utilized high-throughput chemical-genetic screens to uncover a potent class of MET mediators. With the use of in vitro and in vivo models of TNBC, we showed that changing the malignant cell state to a differentiated phenotype by inducing MET reduced mammosphere formation, increased chemosensitivity, and decreased the tumor burden in NSG mice. Delving into the mechanisms of tumor differentiation via ChIP-seq, RNA-seq, and Gene Ontology analysis revealed differences in metabolic status between cell states, which might be exploited in the treatment of TNBC. We also assessed combinations of MET mediators, in order to increase the potency and durability of differentiation. Hence, this study assessed the role of differentiation in the treatment of TNBC and the efficacy of various MET mediators, singly and in combination, in inducing differentiation. Citation Format: Ser Yue Loo, Liping Toh, Elina Pathak, Wilson Tan, Siming Ma, Ju Yuan, Giridharan Periyasamy, Federico Torta, Jack Chan, Tira Tan, Yi Rong Sim, Veronique Tan, Benita Tan, Preetha Madhukumar, Wei Sean Yong, Kong Wee Ong, Chow Yin Wong, Markus R. Wenk, Roger Foo, Yoon-Sim Yap, Elaine Lim, Wai Leong Tam. Inducing cell state transitions in triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B22.

Elucidating therapeutic molecular targets in premenopausal Asian women with recurrent breast cancers

AbstractBreast cancer is an increasing problem in Asia, with a higher proportion of premenopausal patients who are at higher risk of recurrence. Targeted sequencing was performed on DNA extracted from primary tumor specimens of 63 premenopausal Asian patients who relapsed after initial diagnosis of non-metastatic breast cancer. The most prevalent alterations included: TP53 (65%); PIK3CA (32%); GATA3 (29%); ERBB2 (27%); MYC (25%); KMT2C (21%); MCL1 (17%); PRKDC, TPR, BRIP1 (14%); MDM4, PCDH15, PRKAR1A, CDKN1B (13%); CCND1, KMT2D, STK11, and MLH1 (11%). Sixty of the 63 patients (95%) had at least one genetic alteration in a signaling pathway related to cell cycle or p53 signaling. The presence of MCL1 amplification, HIF-1-alpha transcription factor network pathway alterations, and direct p53 effectors pathway alterations were independent predictors of inferior overall survival from initial diagnosis. Comparison with non-Asian premenopausal tumors in The Cancer Genome Atlas (TCGA) revealed a higher prevalence of TP53 mutations among HER2-positive cancers, and more frequent TP53, TET2, and CDK12 mutations among hormone receptor-positive HER2-negative cancers in our cohort. Given the limited number of non-Asian premenopausal breast cancers that had relapsed in TCGA, we compared the frequency of mutations in our cohort with 43 premenopausal specimens from both TCGA and International Cancer Genome Consortium that had relapsed. There was a trend toward higher prevalence of TP53 mutations in our cohort. Certain genomic aberrations may be enriched in tumors of poor-prognosis premenopausal Asian breast cancers. The development of novel therapies targeting these aberrations merit further research.Ethnic diversity: distinct molecular profiles in tumors from premenopausal Asian womenYounger women in Asia with recurrent breast cancer seem to have a higher rate of mutations in the tumor suppressor gene TP53 than do women elsewhere—a finding that could guide drug development in Asia. Yoon-Sim Yap from the
 National Cancer Centre Singapore and coworkers sequenced DNA extracted from the tumor samples of 63 premenopausal women from Singapore and South Korea who relapsed following treatment for non-metastatic breast cancer. The researchers analyzed hundreds of cancer-related genes, and found that the vast majority of women harbored mutations in at least one gene linked to regulating the cell cycle of TP53 signaling. The prevalence of mutations in certain genes, including TP53 itself, was higher than observed previously in non-Asian cohorts, highlighting the need to consider ethnic diversity in genomic studies of breast cancer and in drug development.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype

PurposeThe purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data.MethodsWe identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry.ResultsThere was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11–4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent.ConclusionsThis comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

Abstract 5008: Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting. Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated. Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A “false positive” cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs. Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime. Citation Format: Man Chun Leong, Tse Hui Lim, Chye Ling Tan, Yong Wei Chua, Elaine Hsuen Lim, Kiley Wei Jen Loh, Guek Eng Lee, Rebecca Dent, Raymond Chee Hui Ng, Andrew Wu, Wan-Teck Lim, Alvin Soon-Tiong Lim, Yoon-Sim Yap. Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5008.

Abstract 2668: The impact of CYP2C19 polymorphisms on plasma concentrations and metabolic ratios of tamoxifen and its metabolites in Asian breast cancer patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tamoxifen (TAM) is a prodrug with a complex metabolic pathway involving several metabolic enzymes. Although TAM is mainly metabolised by cytochrome P450 (CYP) 2D6 and CYP3A4/5 enzymes, other CYP enzymes such as CYP1A2, CYP2B6, CYP2C9, and CYP2C19, also catalysed the metabolism of TAM to N-desmethyltamoxifen (NDM), 4-hydroxytamoxifen (4OHT) and endoxifen (END) with 4OHT and END being the active metabolites of TAM. The aim of this study was to investigate the impact of CYP2C19 polymorphisms on the plasma concentrations and metabolic ratios of TAM and its three metabolites, NDM, 4OHT and END. The exons and intron-boundaries of CYP2C19 gene as well as the upstream and downstream regions (about 14 kilobases) were sequenced in 240 Asian healthy subjects (Chinese, Malay, Indians, N=80 each) to identify the polymorphisms present in Asian population. Linkage disequilibrium between identified polymorphisms was examined via Haploview (version 4.0) and tag-SNPs were identified via TAGGER. These tag-SNPs were subsequently genotyped in 164 Asian breast cancer patients using direct sequencing. Plasma levels of TAM and its metabolites were determined at steady state using HPLC with fluorescence detection. Genotypic-phenotypic associations were performed using non-parametric Kruskal-Wallis test and Mann-Whitney U-test. A moderate linkage pattern was observed across the CYP2C19 polymorphisms in the three Asian healthy populations. A total of 13 tag-SNPs and one reported functional SNP were analyzed in Asian breast cancer patients. Patients carrying the AC and CC genotypes of the exonic polymorphism 1251A>C (rs17886522) was associated with 2.3-fold reduction in the median (range) MREND-NDM compared to patients with the reference genotype [AA vs AC+CC: 5.14 (0.92 − 27.81) vs 2.25 (1.26 − 10.72), P = 0.026]. In contrast, median (range) MREND-4-OHT was found to be 1.3-fold higher in patients carrying one or two copies of the −3219T>G variant allele compared to patients carrying the two copies of wild-type allele [TT vs TG + GG: 6.57 (1.99 − 13.18) vs 8.76 (3.78 − 12.80), P = 0.001]. Modest increases in the plasma concentrations of TAM and NDM were associated with 1251A>C (rs17886522). In addition to CYP2D6 polymorphisms, polymorphic variants present in the 5α upstream region of CYP2C19 were found to influence the metabolic ratios of TAM and its metabolites but not the plasma concentrations of the analytes in this exploratory study. The combinative effect of genetic variants in various phase I pharmacogenes on plasma levels of tamoxifen warrants further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2668. doi:1538-7445.AM2012-2668