Genetic Predictors of Chemotherapy-Induced Peripheral Neuropathy from Paclitaxel, Carboplatin and Oxaliplatin: NCCTG/Alliance N08C1, N08CA and N08CB Study.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and potentially permanent adverse effect of chemotherapeutic agents including taxanes such as paclitaxel and platinum-based compounds such as oxaliplatin and carboplatin. Previous studies have suggested that genetics may impact the risk of CIPN. We conducted genome-wide association studies (GWASs) for CIPN in two independent populations who had completed European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ)-CIPN20 assessments (a CIPN-specific 20-item questionnaire which includes three scales that evaluate sensory, autonomic, and motor symptoms). The study population N08Cx included 692 participants from three clinical trials (North Central Cancer Treatment Group (NCCTG) N08C1, N08CA, and N08CB) who had been treated with paclitaxel, paclitaxel plus carboplatin, or oxaliplatin. The primary endpoint for the GWAS was the change from pre-chemotherapy CIPN20 sensory score to the worse score over the following 18 weeks. Study population The Mayo Clinic Breast Disease Registry (MCBDR) consisted of 381 Mayo Clinic Breast Disease Registry enrollees who had been treated with taxane or platinum-based chemotherapy. The primary endpoint for the GWAS assessed was the earliest CIPN20 sensory score available after the completion of chemotherapy. In multivariate model analyses, chemotherapy regimen (p = 3.0 × 10-8) and genetic ancestry (p = 0.007) were significantly associated with CIPN in the N08Cx population. Only age (p = 0.0004) was significantly associated with CIPN in the MCBDR population. The SNP most associated with CIPN was rs56360211 near PDE6C (p =7.92 × 10-8) in N08Cx and rs113807868 near TMEM150C in the MCBDR (p = 1.27 × 10-8). Due to a lack of replication, we cannot conclude that we identified any genetic predictors of CIPN.

Genetic Variations and Health-Related Quality of Life (HRQOL): A Genome-Wide Study Approach.

Health-related quality of life (HRQOL) is an important prognostic patient-reported outcome in oncology. Because prior studies suggest that HRQOL is, in part, heritable, we performed a GWAS to elucidate genetic factors associated with HRQOL in breast cancer survivors. Physical and mental HRQOL were measured via paper surveys that included the PROMIS-10 physical and mental health domain scales in 1442 breast cancer survivors participating in the Mayo Clinic Breast Disease Registry (MCBDR). In multivariable regression analyses, age and financial concerns were significantly associated with global physical health (age: p = 1.6 × 10-23; financial concerns: p = 4.8 × 10-40) and mental health (age: p = 3.5 × 10-7; financial concerns: p = 2.0 × 10-69). Chemotherapy was associated with worse global mental health (p = 0.01). In the GWAS, none of the SNPs reached the genome-wide association significance threshold of 5 × 10-8 for associations with either global physical or global mental health, however, a cluster of SNPs in SCN10A, particularly rs112718371, appeared to be linked to worse global physical health (p = 5.21 × 10-8). Additionally, SNPs in LMX1B, SGCD, PARP12 and SEMA5A were also moderately associated with worse physical and mental health (p < 10-6). These biologically plausible candidate SNPs warrant further study as possible predictors of HRQOL.

A randomized, double-blind, placebo-controlled trial of testosterone for treatment of postmenopausal women with aromatase inhibitor-induced arthralgias: Alliance study A221102.

PURPOSE: To evaluate the efficacy of testosterone supplementation for improving aromatase inhibitor musculoskeletal symptoms (AIMSS). METHODS: Postmenopausal women experiencing moderate-to-severe arthralgias while taking adjuvant aromatase inhibitors for breast cancer were enrolled in this trial. Initially, patients were randomly allocated to receive either a subcutaneous testosterone pellet versus a placebo pellet. Due to slow accrual, the protocol was modified such that additional participants were randomized to receive either a topical testosterone gel or a placebo gel. Changes in patient-reported joint pain were compared between patients receiving testosterone and those receiving placebo using a two-sample t test. Changes in hot flashes and other vasomotor symptoms were also analyzed. Further analyses were conducted to evaluate whether 27 single nucleotide polymorphisms (SNPs) in 14 genes previously associated with AIMSS were associated with testosterone supplementation benefit. RESULTS: While 64% of patients reported an improvement in joint pain at 3 months, there were no significant differences in average pain or joint stiffness at 3 or 6 months between testosterone and placebo arms. Patients receiving testosterone did report improvements in strength, lack of energy, urinary frequency, and stress incontinence (p < 0.05). The subset of patients receiving subcutaneous testosterone also experienced improvements in hot flashes and mood swings. An inherited variant (rs7984870 CC genotype) in TNFSF11 was more likely to be associated with improvements in hot flashes in patients receiving testosterone. CONCLUSION: The doses of testosterone supplementation used in this study did not significantly improve AIMSS. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01573442.

NCCTG N0821 (Alliance): a phase II first-line study of pemetrexed, carboplatin, and bevacizumab in elderly patients with advanced nonsquamous non-small-cell lung cancer with good performance status.

BACKGROUND: We hypothesized that the combination of bevacizumab, carboplatin, and pemetrexed will be an effective first-line regimen in fit, elderly patients with nonsquamous non-small-cell lung cancer. METHODS: Treatment-naïve, stage IIIB/IV nonsquamous non-small-cell lung cancer patients more than 70 years old with good performance status (Eastern Cooperative Oncology Group performance status 0-1) and adequate organ function were eligible. Carboplatin area under the curve 6, pemetrexed 500 mg/m, and bevacizumab 15 mg/kg were administered on day 1 of each 21-day cycle (up to six cycles) followed by maintenance pemetrexed and bevacizumab. The primary end point of 6-month progression-free survival rate of at least 70% was assessed using a one-stage binomial design. Quality of life (QOL) questionnaires were administered. Polymorphisms in genes encoding relevant proteins (drug targets, transport, and metabolism proteins) were correlated with treatment outcome. RESULTS: Fifty-seven eligible patients were enrolled. Median age was 74.5 years. Median treatment cycles received was 6. The most common grade 3 or higher non-hematologic adverse events were fatigue (26%) and hypertension (11%); 16% had grade 4 neutropenia and 6.5% had grade 4 thrombocytopenia. Three patients experienced grade 3/4 hemorrhagic events (one pulmonary, two gastrointestinal). Primary end point of PFS6 was 60% (95% confidence interval [CI]: 45.9-73%). Median PFS was 7.0 months (95% CI: 5.9-10.1), median overall survival was 13.7 months (95% CI: 9.4-16.8). Polymorphic KDR and VEGFA variants correlated with survival and toxicity, respectively. There was no significant change in overall QOL scores over time. CONCLUSION: This regimen is feasible and did not decrease the QOL in this study population. However, it did not meet the primary efficacy end point.

Phase II study of perioperative chemotherapy with cisplatin and pemetrexed in non-small-cell lung cancer.

INTRODUCTION: Pathologic complete response (pCR) with neoadjuvant chemotherapy is associated with improved survival in many solid tumors. We evaluated pCR rate of cisplatin with pemetrexed in non-small-cell lung cancer. METHODS: Patients with stages IB to IIIA non-small-cell lung cancer, Eastern Cooperative Oncology Group performance status 0 to 1 were enrolled in this single-arm phase II trial using two-stage design with 90% power to detect pCR rate of more than or equal to 10%. Pretreatment mediastinal lymph node biopsy was required. Patients received three cycles of cisplatin 75 mg/m with pemetrexed 500 mg/m (day 1 every 21 days) preoperatively and additional two cycles within 60 to 80 days after surgery. The primary end point was pCR. Polymorphisms in FPGS, GGH, SLC19A1, and TYMS genes were correlated with treatment outcomes. RESULTS: Thirty-eight patients were enrolled, with median age of 62.5 years. Preoperatively, 26% had squamous histology, and 34% had biopsy-proven N2 involvement. R0 resection was achieved in 94% of the 34 patients who underwent surgery, and 54% had documented N2 clearance. There was no pCR seen. Median disease-free survival (DFS) and overall survival of these patients have not yet been reached in contrast to median of 13.8 and 24.2 months, respectively, in patients with persistent N2 disease (p = 0.3241 and p = 0.1022, respectively). There was a statistically significant association between DFS and postoperative tumor, node, metastasis stage (p = 0.0429), SLC19A1 rs3788189 TT genotype (p = 0.0821), and viable tumor defined as less than or equal to 10% of resected specimen (p = 0.026). CONCLUSION: The primary end point was not met. Patients with N2 clearance, less than or equal to 10% viable tumor in the resected specimen, and SLC19A1 rs3788189 TT genotype have favorable DFS outcomes.

Low 25(OH) vitamin D3 levels are associated with adverse outcome in newly diagnosed, intensively treated adult acute myeloid leukemia.

BACKGROUND: Several studies have suggested that low 25(OH) vitamin D3 levels may be prognostic in some malignancies, but no studies have evaluated their impact on treatment outcome in patients with acute myeloid leukemia (AML). METHODS: Vitamin D levels were evaluated in 97 consecutive, newly diagnosed, intensively treated patients with AML. MicroRNA expression profiles and single nucleotide polymorphisms (SNPs) in the 25(OH) vitamin D3 pathway genes were evaluated and correlated with 25(OH) vitamin D3 levels and treatment outcome. RESULTS: Thirty-four patients (35%) had normal 25(OH) vitamin D3 levels (32-100 ng/mL), 34 patients (35%) had insufficient levels (20-31.9 ng/mL), and 29 patients (30%) had deficient levels (<20 ng/mL). Insufficient/deficient 25(OH) vitamin D3 levels were associated with worse relapse-free survival (RFS) compared with normal vitamin D3 levels. In multivariate analyses, deficient 25(OH) vitamin D3 , smoking, European Leukemia Network genetic group, and white blood cell count retained their statistical significance for RFS. Several microRNAs and SNPs were associated with 25(OH) vitamin D3 levels, although none remained significant after multiple test corrections; one 25(OH) vitamin D3 receptor SNP, rs10783219, was associated with a lower complete remission rate (P = .0442) and with shorter RFS (P = .0058) and overall survival (P = .0011). CONCLUSIONS: It remains to be determined what role microRNA and SNP profiles play in contributing to low 25(OH) vitamin D3 level and/or outcome and whether supplementation will improve outcomes for patients with AML.

Serum vitamin D metabolites in colorectal cancer patients receiving cholecalciferol supplementation: correlation with polymorphisms in the vitamin D genes.

Cholecalciferol (D(3)) supplementation results in variable increases in serum 25(OH)D(3) levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D(3), 24,25(OH)(2)D(3), 1,25(OH)2D(3) and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D(3) supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D(3) supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D(3) metabolites levels before and after D(3) supplementation was analyzed. The mean baseline serum 25(OH)D(3) level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D(3) supplementation, serum levels of 25(OH)D(3) increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)(2)D(3), 1,25(OH)(2)D(3), VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D(3) and 1,25(OH)(2)D(3) levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D(3) and 1,25(OH)(2)D(3) levels both before and after D(3) supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D(3) levels after supplementation but not with baseline 25(OH)D(3). Our results show that D(3) supplementation increased 25(OH)D(3) levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D(3) insufficiency and suboptimal increase in 25(OH)D(3) levels after D(3) supplementation. Individuals with these genotypes may require higher D(3) supplementation doses to achieve vitamin D(3) sufficiency.

A randomized phase II study of gemcitabine and carboplatin with or without cediranib as first-line therapy in advanced non-small-cell lung cancer: North Central Cancer Treatment Group Study N0528.

INTRODUCTION: The purpose of this study was to assess the safety and efficacy of gemcitabine and carboplatin with (arm A) or without (arm B) daily oral cediranib as first-line therapy for advanced non-small-cell lung cancer. METHODS: A lead-in phase to determine the tolerability of gemcitabine 1000 mg/m on days 1 and 8, and carboplatin on day 1 at area under curve 5 administered every 21 days with cediranib 45 mg once daily was followed by a 2 (A):1 (B) randomized phase II study. The primary end point was confirmed overall response rate (ORR) with 6-month progression-free survival (PFS6) rate in arm A as secondary end point. Polymorphisms in genes encoding cediranib targets and transport were correlated with treatment outcome. RESULTS: On the basis of the safety assessment, cediranib 30 mg daily was used in the phase II portion. A total of 58 and 29 evaluable patients were accrued to arms A and B. Patients in A experienced more grade 3+ nonhematologic adverse events, 71% versus 45% (p = 0.01). The ORR was 19% (A) versus 20% (B) (p = 1.0). PFS6 in A was 48% (95% confidence interval: 35%-62%), thus meeting the protocol-specified threshold of at least 40%. The median overall survival was 12.0 versus 9.9 months (p = 0.10). FGFR1 rs7012413, FGFR2 rs2912791, and VEGFR3 rs11748431 polymorphisms were significantly associated with decreased overall survival (hazard ratio 2.78-5.01, p = 0.0002-0.0095). CONCLUSIONS: The trial did not meet its primary end point of ORR but met its secondary end point of PFS6. The combination with cediranib 30 mg daily resulted in increased toxicity. Pharmacogenetic analysis revealed an association of FGFR and VEGFR variants with survival.

Correlation between polymorphisms of the reduced folate carrier gene (SLC19A1) and survival after pemetrexed-based therapy in non-small cell lung cancer: a North Central Cancer Treatment Group-based exploratory study.

PURPOSE: To correlate polymorphisms in genes involved in the transport, activation, and inactivation of pemetrexed with the outcome of patients with advanced non-small cell lung cancer (NSCLC) treated with pemetrexed. EXPERIMENTAL DESIGN: Data from a phase II NSCLC trial evaluating the optimal schedule of gemcitabine and pemetrexed were used. All patients with available DNA were genotyped for polymorphisms in FPGS, GGH, and SLC19A1 genes. Patients with various genotypes were compared for efficacy and adverse events resulting from pemetrexed. RESULTS: Fifty-four patients had genotype results for all polymorphisms studied. Patients with the homozygous variant genotypes for SLC19A1 IVS4(2117) C>T, IVS5(9148) C>A, and wild-type genotype for exon6(2522) C>T had a significantly better overall survival compared with their counterparts (median overall survival in months: 8.9 [CC] versus 14.0 [CT] versus 16.7 [TT]; 9.4 [CC] versus 10.3 [CA] versus 22.7 [AA]; and 22.7 [CC] versus 10.3 [CT] versus 9.4 [TT] respectively; all log rank p = 0.03). Patients with the heterozygous TC genotype for GGH IVS5(1042) T>C had greater rates of confirmed response + stable disease compared with the TT genotype (85% versus 60%; odds ratio = 4.0; p = 0.06). A greater risk for grade 3/4 SGPT (ALT) elevation was observed in patients heterozygous (GA) for the FPGS IVS1 (28) G>A polymorphism compared with the GG genotype (43% versus 13%; odds ratio = 5.0, p = 0.07). All results were largely consistent within patients with nonsquamous (n = 40) histology. CONCLUSION: Polymorphisms in SLC1A91 seem to predict for survival differences in pemetrexed-treated NSCLC. Additionally, polymorphisms in GGH and FPGS have marginal associations with response and adverse event. These results should be validated in larger prospective studies using pemetrexed.

Phase II trial of pemetrexed plus bevacizumab for second-line therapy of patients with advanced non-small-cell lung cancer: NCCTG and SWOG study N0426.

PURPOSE: To evaluate the efficacy and toxicity of pemetrexed combined with bevacizumab as second-line therapy for patients with advanced non-small-cell lung cancer (NSCLC) and to correlate allelic variants in pemetrexed-metabolizing genes with clinical outcome. PATIENTS AND METHODS: Patients with previously treated NSCLC received pemetrexed (500 mg/m(2) intravenous) combined with bevacizumab (15 mg/kg intravenous) every 3 weeks. The primary end point, evaluated using a one-stage Fleming design for detecting a true success rate of at least 70%, was the proportion of patients who were progression free and on treatment at 3 months. Polymorphisms in genes responsible for pemetrexed transport (reduced folate carrier [SLC19A1]) and metabolism (folylpolyglutamate synthase [FPGS] and gamma-glutamyl hydrolase [GGH]) evaluated in germline DNA (blood) were correlated with treatment outcome. RESULTS: Forty-eight evaluable patients (14 females and 34 males) received a median of four cycles (range, one to 20 cycles). The most common grade 3 or 4 nonhematologic adverse events (AEs) were fatigue (13%), dyspnea (10%), and thrombosis (10%). Grade 3 or 4 hematologic AEs were neutropenia (19%) and lymphopenia (13%). Twenty-four (57%; 95% CI, 41% to 72%) of the first 42 patients met the success criteria. Median overall survival (OS) and progression-free survival (PFS) times were 8.6 and 4.0 months, respectively. The exon 6 (2522)C-->T polymorphism in SLC19A1 correlated with 3-month progression-free status (P = .01) and with PFS (P = .05). The IVS1(1307)C-->T polymorphism in GGH correlated with OS (P = .04). CONCLUSION: The study did not meet its primary end point. However, the median PFS time of 4 months is promising. Pharmacogenetic studies in larger cohorts are needed to definitively identify polymorphisms that predict for survival and toxicity of pemetrexed.

Structural basis of substrate recognition in thiopurine s-methyltransferase.

Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl- l-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl- l-homocysteine and as a ternary complex with S-adenosyl- l-homocysteine and the substrate 6-mercaptopurine, to 1.8 and 2.0 A resolution, respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S N2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases V max and increases K m for 6-mercaptopurine but not K m for S-adenosyl- l-methionine. Substitution at this position with alanine or histidine and similar substitutions of Arg226 (Arg221 in murine TPMT) result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased V max and increased K m for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure.

Interindividual variability in acetaminophen sulfation by human fetal liver: implications for pharmacogenetic investigations of drug-induced birth defects.

BACKGROUND: Acetaminophen (APAP) use in early pregnancy has been associated with the risk of gastroschisis, a rare but serious congenital defect of the abdominal wall. The purpose of this study was to characterize the variability of APAP sulfation in a panel of human fetal livers and to identify the sulfotransferases (SULT) isoform(s) responsible for catalyzing that activity. METHODS: APAP sulfation was determined in a panel of human fetal (n = 73) and postnatal (n = 18) liver cytosol preparations and correlated with the catalytic activity of various SULT isoforms as determined using prototypic substrates and specific antibodies. RESULTS: Of 10 heterologously expressed SULT isoforms examined, SULT1A1, SULT1A3/4, SULT1E1, and SULT2A1 all catalyzed the formation of APAP sulfate with K(m) values of 2.4, 1.5, 1.9, and 3.7 mM, respectively. Catalytic activities for these four isoforms were expressed at varying levels in human fetal liver, and APAP sulfation was positively correlated with each of the four prototypic activities. Several regression and clustering approaches revealed that SULT1A3/4 was the primary determinant of prenatal APAP sulfation but that SULT1A1 or SULT1E1 were also major contributors in subsets of samples. CONCLUSIONS: The results of this study lead to the hypothesis that genetic variation in SULT1A3/4 represents a risk factor for the development of gastroschisis in the offspring of mothers exposed to APAP early in pregnancy. Interpretation of genetic association studies conducted to test this hypothesis will be complicated by the variable contributions of other SULTs toward APAP-sulfate formation in individual subjects.

Effect of resveratrol on 17beta-estradiol sulfation by human hepatic and jejunal S9 and recombinant sulfotransferase 1E1.

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4'-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17beta-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3-20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 microM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated K(i) values of 1.1 microM (liver), 0.6 microM (jejunum), and 2.3 microM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

Identification and characterization of genetic variation in the folylpolyglutamate synthase gene.

Folylpolyglutamate synthase (FPGS) catalyzes the polyglutamation of folic acid, methotrexate, and pemetrexed to produce highly active metabolites. To characterize genetic variation in the FPGS gene, FPGS, have resequenced the gene in four different ethnic populations. Thirty-four single nucleotide polymorphisms were identified including five nonsynonymous coding single nucleotide polymorphisms that altered the FPGS protein sequence: F13L and V22I polymorphisms in the mitochondrial isoform of FPGS, and R466/424C, A489/447V, and S499/457F polymorphisms, which exist in both the mitochondrial and cytosolic isoforms. When expressed in AuxB1 cells, the A447V cytosolic variant was functionally similar to the wild-type cytosolic (WT Cyt) allozyme, whereas the R424C and S457F cytosolic variants were reduced by approximately 2-fold in protein expression compared with WT Cyt (P < 0.01). The intrinsic clearance of glutamate was reduced by 12.3-fold (R424C, P < 0.01) and 6.2-fold (S457F, P < 0.01), whereas the intrinsic clearance of methotrexate was reduced by 4.2-fold (R424C, P < 0.05) and 5.4-fold (S457F, P < 0.05) in these two cytosolic variants when compared with the WT Cyt isoform. Additionally, the in vitro enzyme velocity at saturating pemetrexed concentrations was reduced by 1.6-fold (R424C, P < 0.05) and 2.6-fold (S457F, P < 0.01) compared with WT Cyt. AuxB1 cells harboring these same cytosolic variant allozymes displayed significant increases in the EC(50) for folic acid and in the IC(50) values for both methotrexate and pemetrexed relative to the WT Cyt form of FPGS. These observations suggest that genetic variations in FPGS may alter the efficacy of antifolate therapy in cancer patients.

Human SULT1A1 gene: copy number differences and functional implications.

SULT1A1, which catalyzes the sulfate conjugation of a wide variety of natural and synthetic compounds, is genetically polymorphic. Biochemical and pharmacogenetic studies have demonstrated that individual variation in the level of enzyme activity is inherited. Common single-nucleotide polymorphisms (SNPs) located in the open reading frame and in the 5'-flanking region (5'-FR) may account for a portion of this individual variation. In this study, we demonstrate the presence of SULT1A1 gene deletions and duplications, representing an additional source of variability in the metabolic activity of this enzyme. A quantitative multiplex PCR assay was used to measure the extent of copy number differences and the frequency of these events in different populations. An analysis of DNA from 362 Caucasian-American and 99 African-American showed the presence of 1 to approximately 5 copies of SULT1A1 in individual samples: 5% of Caucasian subjects contained a single copy of the gene and 26% had three or more copies, while 63% of African-American subjects had three or more copies. Analysis of the genomic region surrounding the SULT1A1 gene in three separate cases with a deletion demonstrated that the entire SULT1A1 gene was affected. Reporter assays, constructed for each of the various 5'-FR SNP haplotypes, suggest that these may also play a role in SULT1A1 activity. However, the variability in the level of enzyme activity among 23 human platelet and 267 human liver samples was best explained by gene copy number differences when all sources of genetic variability were considered (P < 0.0001). Overall, these observations have obvious implications for the effectiveness of SULT1A1 as a drug and hormone metabolizing enzyme and its potential role as a risk factor for disease.

Human aromatase: gene resequencing and functional genomics.

Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we "resequenced" all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3'-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp39Arg, Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, and double variant Arg39Cys264 allozymes showed significant decreases in levels of activity and immunoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg39 allozyme, whereas Met201 displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent K(m) value for Thr364 with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg39Cys264) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.

Estrogen bioactivation, genetic polymorphisms, and ovarian cancer.

Recent experimental evidence has shown that catechol estrogens can be activated through metabolism to form depurinating DNA adducts and thereby initiate cancer. Limited data are available regarding this pathway in epithelial ovarian cancer. We conducted a case-control study of 503 incident epithelial ovarian cancer cases at the Mayo Clinic in Rochester, MN, and Jacksonville, FL, and a 48-county region in North Carolina. Six hundred nine cancer-free controls were frequency matched to the cases on age, race, and residence. After an interview to obtain data on risk factors, a sample of blood was collected for DNA isolation. Subjects were genotyped for seven common single nucleotide polymorphisms in four genes involved in catechol estrogen formation (CYP1A1 and CYP1B1) or conjugation (COMT and SULT1A1). Data were analyzed using logistic regression, stratified by race, and with adjustment for design factors and potential confounders. None of the individual genotypes were significantly associated with ovarian cancer risk. However, an oligogenic model that considered the joint effects of the four candidate genes provided evidence for an association between combinations of these genes and ovarian cancer status (P = 0.015). Although preliminary, this study provides some support for the hypothesis that low-penetrance susceptibility alleles may influence risk of epithelial ovarian cancer.

Human phenylethanolamine N-methyltransferase pharmacogenomics: gene re-sequencing and functional genomics.

Phenylethanolamine N-methyltransferase (PNMT, EC2.1.1.28) catalyzes the N-methylation of norepinephrine to form epinephrine. As a step toward understanding the possible contribution of inheritance to individual variation in PNMT-catalyzed epinephrine formation, we 're-sequenced' the entire human PNMT gene, including the three exons, the introns and approximately 1 kb of the 5'-flanking region (5'-FR), using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Within the 3.5 kb re-sequenced, 18 single nucleotide polymorphisms (SNPs) were observed, including four non-synonymous coding SNPs (cSNPs) that resulted in the following alterations in encoded amino acid sequence: Asn9Ser, Thr98Ala, Arg112Cys and Ala175Thr. When constructs for the non-synonymous cSNPs were transiently expressed in COS-1 cells, the Ala98 allozyme displayed significantly lower levels of both activity and immunoreactive protein (p < 0.002) than did the wild-type (WT) enzyme due, at least in part, to accelerated protein degradation by a proteasome-mediated process. Luciferase reporter gene constructs were also created for the six common PNMT 5'-FR haplotypes observed. Significant differences were observed among haplotypes in their ability to drive transcription. These observations raise the possibility of inherited variation in the ability to form epinephrine from norepinephrine as a result of variant PNMT polymorphisms and haplotypes.

Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer.

PURPOSE: We determined the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLT) of 17-allylamino-17-demethoxygeldanamycin (17-AAG) when infused on days 1, 8, and 15 of a 28-day cycle in advanced solid tumor patients. We also characterized the pharmacokinetics of 17-AAG, its effect on chaperone and client proteins, and whether cytochrome P450 (CYP) 3A5 and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphisms affected 17-AAG disposition or toxicity. PATIENTS AND METHODS: An accelerated titration design was used. Biomarkers were measured in peripheral-blood mononuclear cells (PBMCs) at baseline and on days 1 and 15, and pharmacokinetic analysis was performed on day 1 of cycle 1. CYP3A5*3 and NQO1*2 genotypes were determined and correlated with pharmacokinetics and toxicity. RESULTS: Twenty-one patients received 52 courses at 11 dose levels. DLTs at 431 mg/m(2) were grade 3 bilirubin (n = 1), AST (n = 1), anemia (n = 1), nausea (n = 1), vomiting (n = 1), and myalgias (n = 1). No tumor responses were seen. 17-AAG consistently increased heat shock protein (Hsp) 70 levels in PBMCs. At the MTD, the clearance and half-life (t(1/2)) of 17-AAG were 11.6 L/h/m(2) and 4.15 hours, respectively; whereas the active metabolite 17-aminogeldanamycin had a t(1/2) of 7.63 hours. The CYP3A5*3 and NQO1*2 polymorphisms were not associated with 17-AAG toxicity. The CYP3A5*3 polymorphism was associated with higher 17-AAG clearance. CONCLUSION: The MTD of weekly 17-AAG is 308 mg/m(2). 17-AAG induced Hsp70 in PBMCs, indicating that Hsp90 has been affected. Further evaluation of 17-AAG is ongoing using a twice-weekly regimen, and this schedule of 17-AAG is being tested in combination with chemotherapy.

Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics.

1. Estrogens are used as drugs and estrogen exposure is a risk factor for hormone-dependent diseases such as breast cancer. Sulfate conjugation is an important pathway for estrogen metabolism. The sulfotransferase (SULT) enzyme SULT1E1 has the lowest K(m) values for estrogens and catecholestrogens of the 10 known human SULT isoforms. 2. We previously cloned and characterized the human SULT1E1 cDNA and gene as steps toward pharmacogenetic studies. In the present experiments, we set out to determine whether common, functionally significant genetic polymorphisms might exist for SULT1E1. As a first step, we 'resequenced' the eight SULT1E1 exons and exon-intron splice junctions as well as portions of the 5'-flanking region using DNA from 60 African-American and 60 Caucasian-American subjects. 3. In all, 23 polymorphisms, 22 single nucleotide polymorphisms (SNPs) and one insertion deletion were observed. There were three nonsynonymous coding SNPs (cSNPs) that altered the following encoded amino acids: Asp22Tyr, Ala32Val and Pro253His. Among these, 12 pairs of SNPs were tightly linked. In addition, 12 unambiguous SULT1E1 haplotypes were identified, including six that were common to both populations studied. 4. Transient expression in COS-1 cells of constructs containing the three nonsynonymous cSNPs showed significant decreases in SULT1E1 activity for the Tyr22 and Val32 allozymes, with corresponding decreases in levels of immunoreactive protein. There were no changes in levels of either activity or immunoreactive protein for the His253 allozyme. Apparent K(m) values of the Val32 allozyme for the two cosubstrates for the reaction, 17beta-estradiol and 3'-phosphoadenosine 5'-phosphosulfate, were not significantly different from those of the wild-type enzyme, but there was a two- to three-fold increase in K(m) values for the His253 allozyme and a greater than five-fold increase for the Tyr22 allozyme. 5. These observations raise the possibility that genetically determined variation in SULT1E1-catalyzed estrogen sulfation might contribute to the pathophysiology of estrogen-dependent diseases as well as variation in the biotransformation of exogenously administered estrogens.