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1.  Evaluation of CYP2D6 enzyme activity using a Dextromethorphan Breath Test in Women Receiving Adjuvant Tamoxifen 
Pharmacogenetics and genomics  2015;25(4):157-163.
Background
In tamoxifen-treated patients, breast cancer recurrence differs according to CYP2D6 genotype and endoxifen steady state concentrations (Endx Css). The 13Cdextromethorphan breath test (DM-BT), labeled with 13C at the O-CH3 moiety, measures CYP2D6 enzyme activity. We sought to examine the ability of the DM-BT to identify known CYP2D6 genotypic poor metabolizers and examine the correlation between DMBT and Endx Css.
Methods
DM-BT and tamoxifen pharmacokinetics were obtained at baseline (b), 3 month (3m) and 6 months (6m) following tamoxifen initiation. Potent CYP2D6 inhibitors were prohibited. The correlation between bDM-BT with CYP2D6 genotype and Endx Css was determined. The association between bDM-BT (where values ≤0.9 is an indicator of poor in vivo CYP2D6 metabolism) and Endx Css (using values ≤ 11.2 known to be associated with poorer recurrence free survival) was explored.
Results
91 patients were enrolled and 77 were eligible. CYP2D6 genotype was positively correlated with b, 3m and 6m DMBT (r ranging from 0.457-0. 60 p < 0.001). Both CYP2D6 genotype (r = 0.47; 0.56, p <.0001), and bDM-BT (r=0.60; 0.54; p<.001) were associated with 3m and 6m Endx Css respectively. Seven of 9 patients (78%) with low (≤11.2 nM) 3m Endx Css also had low DM-BT (≤0.9) including 2/2 CYP2D6 PM/PM and 5/5 IM/PM. In contrast, 1 of 48 pts (2%) with a low DM-BT had Endx Css > 11.2 nM.
Conclusions
In patients not taking potent CYP2D6 inhibitors, DM-BT was associated with CYP2D6 genotype and 3m and 6 m Endx Css but did not provide better discrimination of Endx Css compared to CYP2D6 genotype alone. Further studies are needed to identify additional factors which alter Endx Css.
doi:10.1097/FPC.0000000000000121
PMCID: PMC4356640  PMID: 25714002
Tamoxifen; CYP2D6; 13C-dextromethorphan breath test (DM-BT)
2.  Loss of Heterozygosity at the CYP2D6 Locus in Breast Cancer: Implications for Germline Pharmacogenetic Studies 
Background:
Controversy exists regarding the impact of CYP2D6 genotype on tamoxifen responsiveness. We examined loss of heterozygosity (LOH) at the CYP2D6 locus and determined its impact on genotyping error when tumor tissue is used as a DNA source.
Methods:
Genomic tumor data from the adjuvant and metastatic settings (The Cancer Genome Atlas [TCGA] and Foundation Medicine [FM]) were analyzed to characterize the impact of CYP2D6 copy number alterations (CNAs) and LOH on Hardy Weinberg equilibrium (HWE). Additionally, we analyzed CYP2D6 *4 genotype from formalin-fixed paraffin-embedded (FFPE) tumor blocks containing nonmalignant tissue and buccal (germline) samples from patients on the North Central Cancer Treatment Group (NCCTG) 89-30-52 tamoxifen trial. All statistical tests were two-sided.
Results:
In TCGA samples (n =627), the CYP2D6 LOH rate was similar in estrogen receptor (ER)–positive (41.2%) and ER-negative (35.2%) but lower in HER2-positive tumors (15.1%) (P < .001). In FM ER+ samples (n = 290), similar LOH rates were observed (40.8%). In 190 NCCTG samples, the agreement between CYP2D6 genotypes derived from FFPE tumors and FFPE tumors containing nonmalignant tissue was moderate (weighted Kappa = 0.74; 95% CI = 0.63 to 0.84). Comparing CYP2D6 genotypes derived from buccal cells to FFPE tumor DNA, CYP2D6*4 genotype was discordant in six of 31(19.4%). In contrast, there was no disagreement between CYP2D6 genotypes derived from buccal cells with FFPE tumors containing nonmalignant tissue.
Conclusions:
LOH at the CYP2D6 locus is common in breast cancer, resulting in potential misclassification of germline CYP2D6 genotypes. Tumor DNA should not be used to determine germline CYP2D6 genotype without sensitive techniques to detect low frequency alleles and quality control procedures appropriate for somatic DNA.
doi:10.1093/jnci/dju401
PMCID: PMC4565524  PMID: 25490892
3.  Comparative Uterotrophic Effects of Endoxifen and Tamoxifen in Ovariectomized Sprague Dawley Rats 
Toxicologic pathology  2014;42(8):1188-1196.
Endoxifen (4-hydroxyl-N-desmethyl tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for three days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was three-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.
doi:10.1177/0192623314525688
PMCID: PMC4177029  PMID: 24670817
endoxifen; tamoxifen; endometrial cell proliferation
4.  Molecular Markers for Novel Therapeutic Strategies in Pancreatic Endocrine Tumors 
Pancreas  2013;42(3):411-421.
Objectives
Pancreatic endocrine tumors (PETs) share numerous features with gastrointestinal neuroendocrine (carcinoid) tumors. Targets of novel therapeutic strategies previously assessed in carcinoid tumors were analyzed in PETs (44 cases).
Methods
Activating mutations in EGFR, KIT, and PDGFRA, and non-response mutations in KRAS, were evaluated. Copy number of EGFR and HER-2/neu was quantified by fluorescence in situ hybridization. Expression of EGFR, PDGFRA, VEGFR1, TGFBR1, Hsp90, SSTR2A, SSTR5, IGF1R, mTOR, and MGMT was measured immunohistochemically.
Results
Elevated EGFR copy number was found in 38% of cases, but no KRAS non-response mutations. VEGFR1, TGFBR1, PDGFRA, SSTR5, SSTR2A, and IGF1R exhibited the highest levels of expression in the largest percentages of PETs.
Anticancer drugs BMS-754807 (selective for IGF1R/IR), 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, targeting Hsp90), and axitinib (directed toward VEGFR1–3/PDGFRA-B/KIT) induced growth inhibition of human QGP-1 PET cells with IC50 values (nM) of 273, 723, and 743, respectively. At growth-inhibiting concentrations, BMS-754807 inhibited IGF1R phosphorylation; 17-AAG induced loss of EGFR, IGF1R, and VEGFR2; and axitinib increased p21Waf1/Cip1(CDKN1A) expression without inhibiting VEGFR2 phosphorylation.
Conclusions
Results encourage further research into multi-drug strategies incorporating inhibitors targeting IGF1R or Hsp90 and into studies of axitinib combined with conventional chemotherapeutics toxic to tumor cells in persistent growth arrest.
doi:10.1097/MPA.0b013e31826cb243
PMCID: PMC3594087  PMID: 23211371
pancreatic endocrine tumors; molecular analysis
5.  Phase I Study of Vorinostat in Combination with Temozolomide in Patients with High-Grade Gliomas: North American Brain Tumor Consortium Study 04-03 
Purpose
A phase I, dose-finding study of vorinostat in combination with temozolomide (TMZ) was conducted to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics in patients with high-grade glioma (HGG).
Experimental Design
This phase I, dose-finding, investigational study was conducted in two parts. Part 1 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day × 5 days every 28 days. Part 2 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day × 5 days of the first cycle and 200 mg/m2/day × 5 days of the subsequent 28-day cycles.
Results
In Part 1, the MTD of vorinostat administered on days 1-7 and 15-21 of every 28 day cycle in combination with TMZ was 500 mg daily. Dose-limiting toxicities (DLTs) included grade 3 anorexia, grade 3 ALT, and grade 5 hemorrhage in the setting of grade 4 thrombocytopenia. In Part 2, the MTD of vorinostat on days 1-7 and 15-21 of every 28 day cycle combined with TMZ was 400 mg daily. No DLTs were encountered, but vorinostat dosing could not be escalated further due to thrombocytopenia. The most common serious adverse events were fatigue, lymphopenia, thrombocytopenia, and thromboembolic events. There were no apparent pharmacokinetic interactions between vorinostat and TMZ. Vorinostat treatment resulted in hyperacetylation of histones H3 and H4 in peripheral mononuclear cells.
Conclusion
Vorinostat in combination with temozolomide is well-tolerated in patients with HGG. A phase I/II trial of vorinostat with radiotherapy and concomitant TMZ in newly diagnosed glioblastoma is underway.
doi:10.1158/1078-0432.CCR-12-1841
PMCID: PMC3947570  PMID: 22923449
High-grade glioma; Temozolomide; Vorinostat; HDAC Inhibitor
6.  UGT1A1 Genotype-guided Phase I Study of Irinotecan, Oxaliplatin, and Capecitabine 
Investigational new drugs  2013;31(6):10.1007/s10637-013-0034-9.
Purpose
We performed a UGT1A1 genotype-guided study to determine the maximum tolerated dose (MTD) and evaluate the toxicities and pharmacokinetics of the combination of capecitabine (CAP), oxaliplatin (OX), and irinotecan (IRIN).
Experimental Design
Patients were screened for UGT1A1 *28 genotype prior to treatment. The starting dose (mg/m2) was IRIN (150), OX (85) and CAP (400), days 2-15. Doses were escalated or de-escalated within each genotype group (*28/*28, *1/*28 and *1/*1). IRIN pharmacokinetics was performed at the MTD.
Results
50 patients were evaluable for toxicity [11 (*28/*28); 18 (*1/*28); 21 (*1/*1)]. UGT1A1 *28/*28 patients experienced hematologic dose limiting toxicity (DLT), requiring dose-de-escalation. The UGT1A1 *28/*28 recommended phase 2 dose (RP2D) was IRIN (75), OX (85), and CAP (400). In contrast, both UGT1A1 *1/*28 and *1/*1 tolerated higher doses of IRIN and non-hematologic toxicity was dose limiting for UGT1A1 *1/*1. The RP2D was IRIN (150), OX (85), and CAP (400) for UGT1A1*1/*28 and IRIN (150), OX (100), and CAP (1600) for UGT1A1 *1/*1. UGT1A1 *1/*28 and *1/*1 patients treated with IRIN (150) had similar AUCs for the active irinotecan metabolite, SN38 (366 +/− 278 and 350 +/− 159 ng/ml*hr, respectively). UGT1A1 *28/*28 patients (n=3) treated with a lower IRIN dose (100) had non-significantly higher mean SN38 exposures (604 +/− 289 ng/ml*hr, p=0.14). Antitumor activity was observed in all genotype groups.
Conclusions
UGT1A1 genotype affects the dose and pharmacokinetics of the CAPIRINOX regimen and UGT1A1 genotype-guided dosing of CAPIRINOX is ongoing in a phase II study of small bowel cancer (NCT00433550).
doi:10.1007/s10637-013-0034-9
PMCID: PMC3880122  PMID: 24114122
Irinotecan; Capecitabine; Oxaliplatin; UGT1A1
7.  A Phase I Trial Of MK-2206 In Children with Refractory Malignancies: A Children's Oncology Group Study 
Pediatric blood & cancer  2014;61(7):1246-1251.
Background
We report results of a phase I trial designed to estimate the maximum tolerated dose (MTD), describe dose-limiting toxicities (DLT), and characterize the pharmacokinetic profile of MK-2206, an AKT inhibitor, in children with refractory or recurrent malignancies.
Procedure
MK-2206 was administered either every other day (schedule 1), or once a week (Schedule 2) in a 28-day cycle. Dose escalations in increments of ∼30% were independently made in each part using the rolling-six design. Serial pharmacokinetic (PK) studies were obtained. Biological studies include analysis of PI3K/PTEN/AKT-cell signaling pathway in pre and post-therapy in PBMC and in tumors at diagnosis or recurrence.
Results
Fifty patients [26 males, median age 12.6 years (range, 3.1-21.9)] with malignant glioma (16), ependymoma (4), hepatocellular carcinoma (3), gliomatosis cereberi (2) or other tumors (22) were enrolled; 40 were fully evaluable for toxicity (schedule 1 n=23; schedule 2 n=17). Schedule 1 DLTs included: grade 3 dehydration in 1/6 patients at 28 mg/m2; grade 4 hyperglycemia and neutropenia in 1/6 patients at 45 mg/ m2; and grade 3 rash in 3/6 patients at dose level 4 (58 mg/m2). Schedule 2 DLTs included: grade 3 alkaline phosphatase in 1/6 patients at 90 mg/m2; grade 3 rash in 1/6 patients at 120 mg/ m2, and grade 3 rash in 2/6 patients at 155 mg/m2.
Conclusions
The recommended pediatric phase 2 dose of MK-2206 is 45 mg/m2/dose every other day or 120 mg/m2/dose weekly. Pharmacokinetics appeared linear over the dose range studied.
doi:10.1002/pbc.25023
PMCID: PMC4456022  PMID: 24664955
Phase I trial; MK-2206; AKT inhibitor; pediatric; relapsed solid tumors
8.  Mouse pharmacokinetics and metabolism of the curcumin analog, 4-Piperidione,3,5-bis[(2-fluorophenyl)methylene]-acetate(3E,5E) (EF-24; NSC 716993) 
Cancer chemotherapy and pharmacology  2014;73(6):1137-1146.
Purpose
Curcumin, a keto-enol constituent of turmeric, has in vitro and in vivo antitumor activity. However, in vivo potency is low due to poor oral absorption. The mono-carbonyl analog, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone acetate (EF-24, NSC 716993), exhibited broad spectrum activity in the NCI anti-cancer cell line screen and potent anti-angiogenesis activity in a HUVEC cell migration assay. The purpose of this study was to characterize the preclinical pharmacology of EF-24 in mice.
Methods
EF-24 plasma stability, protein binding, pharmacokinetics and metabolism were characterized utilizing an LC/MS/MS assay.
Results
An LC/MS/MS assay that incorporated protein precipitation with methanol, reverse-phase HPLC separation under gradient elution using an aqueous methanol mobile phase containing 0.1% formic acid and positive electrospray ionization detection of the m/z 312 > 149 transition for EF-24. The assay was linear over the range 7.8-1000 nM. Plasma protein binding was > 98% with preferential binding to albumin. EF-24 plasma disposition in mice after i.v. administration of a 10 mg/kg dose was best fit to a 3-compartment open model. The terminal elimination half-life and plasma clearance values were 73.6 min and 0.482 L/min/kg, respectively. EF-24 bioavailability was 60% and 35% after oral and i.p. administration, respectively. NADPH-dependent metabolism of EF-24 loss in liver microsomal preparations yielded several metabolites consistent with EF-24 hydroxylation and reduction.
doi:10.1007/s00280-014-2447-3
PMCID: PMC4392893  PMID: 24760417
EF-24; curcumin; LC/MS/MS; pharmacokinetics; mouse
9.  A Phase I Study of Intermittently Dosed Vorinostat in Combination with Bortezomib in Patients with Advanced Solid Tumors 
Investigational new drugs  2013;32(2):323-329.
Background
Accumulating evidence shows evidence of efficacy with the combination of vorinostat and bortezomib in solid tumors. We previously examined a once-daily continuous dosing schedule of vorinostat in combination with bortezomib which was well tolerated in cycles 1 and 2; however, there was concern regarding the tolerability through multiple cycles. This study was conducted to evaluate an intermittent dosing schedule of vorinostat with bortezomib.
Methods
Vorinostat was initially administered orally twice daily on days 1–14 with bortezomib IV on days 1, 4, 8, and 11 of a 21 day cycle. Two DLTs (elevated ALT and fatigue) were observed at dose level 1, thus the protocol was amended to administer vorinostat intermittently twice daily on days 1–4 and 8–11.
Results
29 patients were enrolled; 13 men and 16 women. Common cancer types included sarcoma, pancreatic, colorectal, GIST, and breast. The most common Grade 3–4 toxicities at any dose level included thrombocytopenia, fatigue, increased ALT, elevated INR, and diarrhea. DLTs in the intermittent dosing scheduled included thrombocytopenia and fatigue. The Cmax and AUC for the intermittent dosing regimen were similar to those observed in the daily dosing. In this heavily pretreated population, stable disease was observed in patients with sarcoma, colorectal adenocarcinoma and GIST.
Conclusions
The MTD was established at vorinostat 300 mg BID on days 1–4 and 8–11 and bortezomib 1.3 mg/m2 IV on days 1, 4, 8, and 11 of a 21 day cycle. Tolerability was not improved with the intermittent dosing schedule of vorinostat when compared to continuous dosing.
doi:10.1007/s10637-013-0035-8
PMCID: PMC3949160  PMID: 24114123
SAHA; vorinostat; PS-341; bortezomib; phase I
10.  Pharmacokinetics of endoxifen and tamoxifen in female mice: implications for comparative in vivo activity studies 
Cancer chemotherapy and pharmacology  2014;74(6):1271-1278.
Background
Reduced CYP2D6 metabolism and low Z-endoxifen (ENDX) concentrations may increase the risk of breast cancer recurrence in tamoxifen (TAM)-treated women. Little is known regarding the differences between TAM and ENDX murine pharmacokinetics or the effect of administration route on plasma concentrations of each drug.
Methods
The pharmacokinetics of TAM and ENDX were characterized in female mice.
Results
For subcutaneous [s.c.] and oral TAM (4, 10 and 20 mg/kg), TAM AUC increased in a linear manner, but concentrations of the active metabolites [ENDX and 4-hydroxytamoxifen (4HT)] remained low. For oral TAM (20 mg), 4HT concentrations were tenfold greater (>25 ng/ml) than achievable in TAM-treated humans. Both oral (10–200 mg/kg) and s.c. (2.5–25 mg/kg) ENDX·HCl resulted in a greater than dose-proportional increase in AUC, with eightfold greater ENDX concentrations than an equivalent TAM dose. ENDX accumulated in plasma after 5-day dosing of 25 or 100 mg/kg ENDX·HCl and exceeded target concentrations of 0.1 and 1.0 μM, respectively, by twofold to fourfold.
Conclusions
In murine models, oral ENDX yields substantially higher ENDX concentrations, compared to TAM. The low 4HT and ENDX concentrations observed in mice receiving s.c. TAM mirror the TAM pharmacokinetics in humans with impaired CYP2D6 metabolism. These data support the ongoing development of ENDX as a novel agent for the endocrine treatment of ER-positive breast cancer.
doi:10.1007/s00280-014-2605-7
PMCID: PMC4343319  PMID: 25318936
Endoxifen; Tamoxifen; Mouse; Pharmacokinetics; Estrogen receptor; MCF7
11.  CYP2D6 Metabolism and Patient Outcome in the Austrian Breast and Colorectal Cancer Study Group Trial (ABCSG) 8 
Background
Controversy exists regarding CYP2D6 genotype and tamoxifen efficacy.
Methods
A matched case-control study was conducted utilizing the Austrian Breast and Colorectal Cancer Study Group Trial 8 that randomized post-menopausal women with estrogen receptor positive breast cancer to tamoxifen for 5 years (Arm A) or tamoxifen for 2 years followed by anastrozole for 3 years (Arm B). Cases had disease recurrence, contralateral breast cancer, second non-breast cancer, or died. For each case, controls were identified from the same treatment arm of similar age, surgery/radiation, and TNM stage. Genotyping was performed for alleles associated with no (PM; *3, *4, *6); reduced (IM; *10, and *41); and extensive (EM: absence of these alleles) CYP2D6 metabolism.
Findings
The common CYP2D6 *4 allele was in Hardy Weinberg Equilibrium. In Arm A during the first 5 years of therapy, women with 2 poor alleles (PM/PM: OR=2.45, 95% CI: 1.05–5.73, p=0.04) and women with one poor allele (PM/IM or PM/EM: OR=1.67, 95% CI: 0.95–2.93, p=0.07) had a higher likelihood of an event than women with two extensive alleles (EM/EM). In years 3–5 when patients remained on tamoxifen (Arm A) or switched to anastrozole (Arm B), PM/PM tended towards a higher likelihood of a disease event relative to EM/EM (OR= 2.40, 95% CI: 0.86–6.66, p=0.09) among women on Arm A but not among women on Arm B (OR= 0.28; 95% CI: 0.03–2.30).
Conclusion
In ABCSG8, the negative effects of reduced CYP2D6 metabolism were observed only during the period of tamoxifen administration, and not after switching to anastrozole.
doi:10.1158/1078-0432.CCR-12-2153
PMCID: PMC3548984  PMID: 23213055
Tamoxifen; CYP2D6; metabolism; anastrozole; breast cancer; estrogen receptor
12.  A Phase I Study of Vorinostat in Combination with Bortezomib in Patients with Advanced Malignancies 
Investigational new drugs  2013;31(6):1539-1546.
Background
A phase I study to assess the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), pharmacokinetics (PK) and antitumor activity of vorinostat in combination with bortezomib in patients with advanced solid tumors.
Methods
Patients received vorinostat orally once daily on days 1–14 and bortezomib intravenously on days 1, 4, 8 and 11 of a 21-day cycle. Starting dose (level 1) was vorinostat (400 mg) and bortezomib (0.7 mg/m2). Bortezomib dosing was increased using a standard phase I dose-escalation schema. PKs were evaluated during cycle 1.
Results
Twenty-three patients received 57 cycles of treatment on four dose levels ranging from bortezomib 0.7 mg/m2 to 1.5 mg/m2. The MTD was established at vorinostat 400 mg daily and bortezomib 1.3 mg/m2. DLTs consisted of grade 3 fatigue in three patients (1 mg/m2,1.3 mg/m2 and 1.5 mg/m2) and grade 3 hyponatremia in one patient (1.5 mg/m2). The most common grade 1/2 toxicities included nausea (60.9%), fatigue (34.8%), diaphoresis (34.8%), anorexia (30.4%) and constipation (26.1%). Objective partial responses were observed in one patient with NSCLC and in one patient with treatment-refractory soft tissue sarcoma. Bortezomib did not affect the PKs of vorinostat; however, the Cmax and AUC of the acid metabolite were significantly increased on day 2 compared with day 1.
Conclusions
This combination was generally well-tolerated at doses that achieved clinical benefit. The MTD was established at vorinostat 400 mg daily x 14 days and bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 of a 21-day cycle.
doi:10.1007/s10637-013-0029-6
PMCID: PMC3901262  PMID: 24114121
SAHA; vorinostat; PS-341; bortezomib; phase I
13.  Gemcitabine Cytotoxicity: Interaction of Efflux and Deamination 
Gemcitabine is a cytidine analogue used in the treatment of various solid tumors. Little is known about how gemcitabine and its metabolites are transported out of cells. We set out to study the efflux of gemcitabine and the possible consequences of that process in cancer cells. We observed the efflux of gemcitabine and its deaminated metabolite, 2’,2’-difluorodeoxyuridine (dFdU) using high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) after gemcitabine treatment. Non-selective ABCC-transport inhibition with probenecid significantly increased intracellular dFdU concentrations, with a similar trend observed with verapamil, a non-selective ABCB1 and ABCG2 transport inhibitor. Neither probenecid nor verapamil altered intracellular gemcitabine levels after the inhibition of deamination with tetrahydrourudine, suggesting that efflux of dFdU, but not gemcitabine, was mediated by ABC transporters. MTS assays showed that probenecid increased sensitivity to gemcitabine. While dFdU displayed little cytotoxicity, intracellular dFdU accumulation inhibited cytidine deaminase, resulting in increased gemcitabine levels and enhanced cytotoxicity. Knockdown of ABCC3, ABCC5 or ABCC10 individually did not significantly increase gemcitabine sensitivity, suggesting the involvement of multiple transporters. In summary, ABCC-mediated efflux may contribute to gemcitabine resistance through increased dFdU efflux that allows for the continuation of gemcitabine deamination. Reversing efflux-mediated gemcitabine resistance may require broad-based efflux inhibition.
doi:10.4172/2157-7609.1000107
PMCID: PMC3144579  PMID: 21804948
Gemcitabine; Cytotoxicity; Drug Efflux; Transport
14.  A Pediatric Phase 1 Trial Of Vorinostat And Temozolomide In Relapsed Or Refractory Primary Brain Or Spinal Cord Tumors: A Children’s Oncology Group Phase 1 Consortium Study 
Pediatric blood & cancer  2013;60(9):1452-1457.
Purpose
We conducted a pediatric phase I study to estimate the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetic properties of vorinostat, a histone deacetylase (HDAC) inhibitor, when given in combination with temozolomide in children with refractory or recurrent CNS malignancies.
Patients and Methods
Vorinostat, followed by temozolomide approximately one hour later, was orally administered, once daily, for 5 consecutive days every 28 days at 3 dose levels using the rolling 6 design. Studies of histone accumulation in peripheral blood mononuclear cells were performed on day 1 at 0, 6, and 24 h after vorinostat dosing. Vorinostat pharmacokinetics (PK) and serum MGMT promoter status were also assessed
Results
Nineteen eligible patients were enrolled and eighteen patients were evaluable for toxicity. There were no DLTs observed at dose level 1 or 2. DLTs occurred in 4 patients at dose level 3: thrombocytopenia (4), neutropenia (3), and leucopenia (1). Non-dose limiting grade 3 or 4 toxicities related to protocol therapy were also hematologic and included neutropenia, lymphopenia, thrombocytopenia, anemia, and leucopenia. Three patients exhibited stable disease and one patient had a partial response. There was no clear relationship between vorinostat dosage and drug exposure over the dose range studied. Accumulation of acetylated H3 histone in PBMC was observed after administration of vorinostat.
Conclusion
Five-day cycles of vorinostat in combination with temozolomide are well tolerated in children with recurrent CNS malignancies with myelosuppression as the DLT. The recommended phase II combination doses are vorinostat, 300 mg/m2/day and temozolomide,150 mg/m2/day.
doi:10.1002/pbc.24541
PMCID: PMC4139006  PMID: 23554030
vorinostat; temozolomide phase I trial; pediatric cancer; Children’s Oncology Group
15.  Gemcitabine Pharmacogenomics: Deoxycytidine Kinase (DCK) and Cytidylate Kinase (CMPK) Gene Resequencing and Functional Genomics 
Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK, and from 78 to 112% of WT for CMPK – with no significant differences in apparent Km values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (Rp = 0.89, P = 0.0004) but not for CMPK (Rp = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variation in DCK and CMPK and systematically evaluated its functional implications. These gene sequence differences may contribute to variation in the metabolic activation of gemcitabine and other cytidine antimetabolites.
doi:10.1124/dmd.108.020925
PMCID: PMC3066084  PMID: 18556440
16.  Selective Estrogen Receptor Modulators and Pharmacogenomic Variation in ZNF423 Regulation of BRCA1 Expression: Individualized Breast Cancer Prevention 
Cancer discovery  2013;3(7):812-825.
The selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high risk women by 50%, but this FDA-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 (SNPs) 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 odds ratio 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.
doi:10.1158/2159-8290.CD-13-0038
PMCID: PMC3710533  PMID: 23764426
tamoxifen; raloxifene; breast cancer prevention; ZNF423; CTSO; BRCA1; single nucleotide polymorphisms; genome-wide association study
17.  Evaluation of CYP2D6 and Efficacy of Tamoxifen and Raloxifene in Women Treated for Breast Cancer Chemoprevention: Results from the NSABP P-1 and P-2 Clinical Trials 
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 performed a nested case-control study in the context of the NSABP P-1 and P-2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, as well as metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both non-invasive and invasive) while on five years of SERM therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was performed for alleles associated with absent (*3, *4, *5, *6), reduced (*10, *17, *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded.
Results
591 cases were matched to 1126 controls and DNA was genotyped in >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.
Conclusions
In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy.
doi:10.1158/1078-0432.CCR-11-0860
PMCID: PMC3207003  PMID: 21880792
tamoxifen; breast cancer; prevention; CYP2D6; polymorphism
18.  Probable fatal drug interaction between intravenous fenretinide, ceftriaxone, and acetaminophen: a case report from a New Approaches to Neuroblastoma (NANT) Phase I study 
BMC Research Notes  2014;7:256.
Background
Patients with relapsed/refractory stage 4 high-risk neuroblastoma were enrolled on a phase I study (NANT2004-03) of intravenous fenretinide emulsion. Pharmacokinetic samples were collected during and after the infusion, and the levels were measured using an HPLC system. A likely case of a fatal drug interaction between fenretinide, ceftriaxone, and acetaminophen is described, including the pharmacokinetics of fenretinide, laboratory data, and post-mortem autopsy in a pediatric neuroblastoma patient treated on this study.
Case presentation
On Day 4 of a scheduled 5-day-infusion of intravenous fenretinide, the patient developed a fever, acetaminophen was started, ceftriaxone initiated for possible bacteremia, and fenretinide level doubled from 56 to 110 μM. Over the next three days, although blood cultures remained negative, the patient’s condition deteriorated rapidly. Acute liver failure was diagnosed on Day 7, and the patient expired on Day 20 of fulminant hepatic failure with associated renal, cardiac, and hemorrhagic/coagulation toxicities. Autopsy showed extensive hemorrhagic necrosis of the liver, marked bile duct proliferation, and abundant hemosiderin, consistent with cholestasis and drug toxicity.
Conclusions
After extensive review of patient data, the clinical course, and the literature, we conclude that observed hepatic toxicity was likely due to a drug interaction between fenretinide and concomitant ceftriaxone and acetaminophen. None of the other 16 patients treated on this study experienced significant hepatic toxicity. Although the prevalence of cholestasis with ceftriaxone usage is relatively high, the potential drug interaction with these concomitant medications has not been previously reported. Concomitant use of fenretinide, ceftriaxone, and acetaminophen should be avoided.
doi:10.1186/1756-0500-7-256
PMCID: PMC4006848  PMID: 24755475
Ceftriaxone; Fenretinide; Acetaminophen; Drug interaction; Biliary sludge; Fulminant hepatic failure
19.  A Phase I Trial of Vorinostat and Alvocidib in Patients with Relapsed, Refractory or Poor Prognosis Acute Leukemia, or Refractory Anemia with Excess Blasts-2 
Purpose
This phase I study was conducted to identify the MTD of alvocidib when combined vorinostat in patients with relapsed, refractory, or poor prognosis acute leukemia, or refractory anemia with excess blasts-2 (RAEB-2). Secondary objectives included investigating the pharmacokinetic and pharmacodynamic effects of the combination.
Experimental Design
Patients received vorinostat (200 mg orally, 3 times a day [TID], for 14 days), on a 21-day cycle, combined with 2 different alvocidib administration schedules: a 1-h intravenous infusion, daily x 5; or a 30-min loading infusion followed by a 4-h maintenance infusion, weekly x 2. The alvocidib dose was escalated using a standard 3+3 design.
Results
Twenty-eight patients were enrolled and treated. The alvocidib MTD was 20 mg/m2 (30-min loading infusion) followed by 20 mg/m2 (4-h maintenance infusion) on days 1 and 8, in combination with vorinostat. The most frequently encountered toxicities were cytopenias, fatigue, hyperglycemia, hypokalemia, hypophosphatemia, and QT prolongation. Dose limiting toxicities (DLTs) were cardiac arrhythmia-atrial fibrillation and QT prolongation. No objective responses were achieved, although 13 of 26 evaluable patients exhibited stable disease. Alvocidib appeared to alter vorinostat pharmacokinetics, whereas alvocidib pharmacokinetics were unaffected by vorinostat. Ex vivo exposure of leukemia cells to plasma obtained from patients after alvocidib treatment blocked vorinostat-mediated p21CIP1 induction and down-regulated Mcl-1 and p-RNA Pol II for some specimens, although parallel in vivo bone marrow responses were infrequent.
Conclusions
Alvocidib combined with vorinostat is well tolerated. Although disease stabilization occurred in some heavily pretreated patients, objective responses were not obtained with these schedules.
doi:10.1158/1078-0432.CCR-12-2926
PMCID: PMC3618599  PMID: 23515411
Vorinostat; Alvocidib; Acute Leukemia; Clinical Trial; Phase I
20.  CYP2D6 and HOXB13/IL17BR: Combining inherited and tumor gene markers for prediction of tamoxifen resistance 
Purpose
Genetic variation in Cytochrome P450 2D6 (CYP2D6) and the gene expression ratio of the homeobox 13 (HOXB13) to interleukin-17B receptor (IL-17BR) are associated with tamoxifen resistance. We sought to determine the combined effect of inherited (CYP2D6) and somatic (HOXB13/IL17BR) gene variation in tamoxifen treated breast cancer.
Experimental Design
Retrospective analysis of women with node negative breast cancer randomized to receive 5 years of tamoxifen (NCCTG 89-30-52). CYP2D6 metabolism (extensive or decreased) was based on CYP2D6*4 genotype and presence/absence of a CYP2D6 inhibitor. RT-PCR profiles for HOXB13 and IL-17BR and the cut-point separating patients into high and low risk categories according to disease-free survival (DFS) were utilized. A risk factor (CYP2D6:HOXB13/IL17BR) representing the four categories of combining CYP2D6 metabolism (extensive or decreased) and HOXB13/IL17BR (low or high) was created. The association between CYP2D6:HOXB13/IL17BR and DFS and overall survival (OS) was assessed using the log-rank test and proportional hazards modeling.
Results
CYP2D6 metabolism and HOXB13/IL17BR gene ratio was available in 110/160 (69%) patients. The combined CYP2D6:HOXB13/IL17BR risk factor was significantly associated with DFS (log rank p=0.004) and OS (p=0.009). Relative to women with extensive CYP2D6 metabolism and low HOXB13/IL17BR, those with either decreased metabolism or a high HOXB13/IL17BR ratio had significantly worse OS [adjusted hazard ratio (HR) =2.41, 95% confidence interval:1.08-5.37; p=0.031) whereas women with both decreased metabolism and high HOXB13/IL17BR had the shortest survival (adjusted HR=3.15, 95% CI:1.17-8.52, p=0.024).
Conclusions
An index comprised of inherited (CYP2D6) and tumor (HOXB13/IL17BR) gene variation identifies patients with varying degrees of resistance to tamoxifen.
doi:10.1158/1078-0432.CCR-08-0619
PMCID: PMC2596658  PMID: 18794098
Tamoxifen; Cytochrome P450 2D6; HOXB13/IL17BR; Breast cancer
21.  A Phase I Trial of Vorinostat and Bortezomib in Children with Refractory or Recurrent Solid Tumors: A Children’s Oncology Group Phase I Consortium Study (ADVL0916) 
Pediatric blood & cancer  2012;60(3):390-395.
Background
A pediatric phase I trial was performed to determine the maximum tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of vorinostat and bortezomib, in patients with solid tumors.
Procedure
Oral vorinostat was administered on days 1–5 and 8–12 of a 21 day cycle (starting dose 180 mg/m2/day with dose escalations to 230 and 300 mg/m2/day). Bortezomib (1.3 mg/m2 i.v.) was administered on days 1, 4, 8, and 11 of the same cycle. PK and correlative biology studies were performed during cycle 1.
Results
Twenty-three eligible patients [17 male, median age 12 years (range, 1–20)] were enrolled of whom 17 were fully evaluable for toxicity. Cycle 1 DLTs that occurred in 2/6 patients at dose level 3 (vorinostat 300 mg/m2/day) were grade 2 sensory neuropathy that progressed to grade 4 (n=1) and grade 3 nausea and anorexia (n=1). No objective responses were observed. There was wide interpatient variability in vorinostat PK parameters. Bortezomib disposition was best described by a three-compartment model that demonstrated rapid distribution followed by prolonged elimination. We did not observe a decrease in NF-κB activity or Grp78 induction after bortezomib treatment in PBMCs from solid tumor patients.
Conclusion
The recommended phase 2 dose and schedule is vorinostat (230 mg/m2/day PO on days 1–5 and 8–12) in combination with bortezomib (1.3 mg/m2/day i.v. on days 1,4, 8, and 11 of a 21 day cycle) in children with recurrent or refractory solid tumors.
doi:10.1002/pbc.24271
PMCID: PMC3511610  PMID: 22887890
vorinostat; bortezomib; phase I trial; pediatric cancer; solid tumors; Children’s Oncology Group
22.  Phase I study of tanespimycin in combination with bortezomib in patients with advanced solid malignancies 
Investigational new drugs  2013;31(5):1251-1256.
Summary
Purpose
To determine the maximum tolerated dose (MTD) and characterize the dose-limiting toxicities (DLT) of tanespimycin when given in combination with bortezomib.
Experimental design
Phase I dose-escalating trial using a standard cohort “3+3” design performed in patients with advanced solid tumors. Patients were given tanespimycin and bortezomib twice weekly for 2 weeks in a 3 week cycle (days 1, 4, 8, 11 every 21 days).
Results
Seventeen patients were enrolled in this study, fifteen were evaluable for toxicity, and nine patients were evaluable for tumor response. The MTD was 250 mg/m2 of tanespimycin and 1.0 mg/m2 of bortezomib when used in combination. DLTs of abdominal pain (13%), complete atrioventricular block (7%), fatigue (7%), encephalopathy (7%), anorexia (7%), hyponatremia (7%), hypoxia (7%), and acidosis (7%) were observed. There were no objective responses. One patient had stable disease.
Conclusions
The recommended phase II dose for twice weekly 17-AAG and PS341 are 250 mg/m2 and 1.0 mg/m2respectively, on days 1, 4, 8 and 11 of a 21 day cycle.
doi:10.1007/s10637-013-9946-7
PMCID: PMC3929968  PMID: 23543109
Phase I Trials; tanespimycin; bortezomib; solid tumors
23.  Association Between CYP2D6 Polymorphisms and Outcomes Among Women With Early Stage Breast Cancer Treated With Tamoxifen 
Context
The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor–positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme.
Objective
To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen.
Design, Setting, and Patients
Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n = 609), heterozygous extensive/intermediate (n = 637), or poor (n = 79) CYP2D6 metabolism.
Main Outcome Measures
Time to recurrence, event-free survival, disease-free survival, and overall survival.
Results
Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and29.0%for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04–1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10–3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06–1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03–1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88–1.51).
Conclusion
Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes.
doi:10.1001/jama.2009.1420
PMCID: PMC3909953  PMID: 19809024
24.  Phase 2 Trial of Pemetrexed in Children and Adolescents with Refractory Solid Tumors: a Children’s Oncology Group Study 
Pediatric blood & cancer  2012;60(2):237-241.
Background
Pemetrexed is a multi-targeted antifolate that inhibits key enzymes involved in nucleotide biosynthesis. We performed a phase 2 trial of pemetrexed in children with refractory or recurrent solid tumors, including CNS tumors, to estimate the response rate and further define its toxicity profile.
Procedure
Pemetrexed, at a dose of 1910 mg/m2, was administered as a 10-minute intravenous infusion every 21 days. Patients also received vitamin B12, daily multivitamin supplementation, and dexamethasone. A two-stage design (10 + 10) was employed in each of the following disease strata: osteosarcoma, Ewing sarcoma/peripheral primitive neuroectodermal tumor (PNET), rhabdomyosarcoma, neuroblastoma, ependymoma, medulloblastoma/supratentorial PNET, and non-brainstem high-grade glioma.
Results
Seventy-two eligible subjects (39 males) were enrolled. Median age was 11 years (range 3–23). Sixty-eight were evaluable for response. The median number of cycles administered was 2 (range 1–13). No complete or partial responses were observed. Stable disease, for a median of 5 (range 4–13) cycles, was observed in 5 patients (ependymoma, Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma; n=1 each). Neutropenia (44%), anemia (35%), and elevated alanine transaminase (35%) attributable to pemetrexed were the most commonly recurring toxicities observed in patients receiving multiple cycles. Other toxicities attributed to pemetrexed occurring in ≥10% of cycles included thrombocytopenia (30%), fatigue (18%), nausea (14), hyperglycemia (13%), rash (11%), vomiting (13%), and hypophosphatemia (11%).
Conclusions
Pemetrexed, administered as an intravenous infusion every 21 days, was tolerable in children and adolescents with refractory solid tumors, including CNS tumors, but did not show evidence of objective anti-tumor activity in the childhood tumors studied.
doi:10.1002/pbc.24244
PMCID: PMC3463652  PMID: 22745043
pemetrexed; phase 2; antifolate
25.  The genomic landscape of small intestine neuroendocrine tumors 
The Journal of Clinical Investigation  2013;123(6):2502-2508.
Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 106 nucleotides (range, 0–0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways.
doi:10.1172/JCI67963
PMCID: PMC3668835  PMID: 23676460

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