Clinical Pharmacogenetics

我们实验室对药物遗传学有浓厚的兴趣。我们一直在积极研究生殖系遗传变异如何改变各种抗癌药物的药代动力学、反应和毒性，从而在已经很窄的治疗窗口中促进临床结果的个体间差异。我们已经建立了这些多态性和它们的表型之间的分子联系，因为它与药物治疗有关。我们的大部分工作集中在药物代谢和转运候选基因的遗传变异上，如ABCB1 (p -糖蛋白，MDR1), ABCG2 (BCRP), SLCO1B3 (OATP1B3, OATP8), CYP3A4, CYP3A5, CYP1B1, CYP2C19, CYP2D6, UGT1A1， UGT1A9等。我们也对非候选基因方法感兴趣，其中探索大量多态性以建立与临床结果的关系，并进行实验以验证探索性扫描产生的潜在致病等位基因。我们已经与Affymetrix合作对DMET芯片进行了beta测试，该芯片包含170种药物处置基因中的1256种遗传变异，目前正在建立一项临床试验，在NCI接受治疗的患者将使用DMET芯片进行基因分型，以探索这些基因与几种癌症的各种治疗之间的潜在联系。我们目前正在验证最初DMET芯片实验的结果。虽然许多这些研究是为了解释一些遗传对药代动力学变异性的影响而进行的，但我们也对阐明几种主要抗癌药物的药效学和治疗结果的遗传标记有浓厚的兴趣，因为这一领域的研究相当少。多西紫杉醇在药代动力学和毒性方面表现出显著的个体差异。沙利度胺是一种有效的抗癌药物，也表现出广泛的药理变异。过去的药物遗传学研究并没有解释这种变异。前列腺癌患者参加了一项随机II期试验，使用多西他赛和沙利度胺与单独使用多西他赛进行基因分型，使用Affymetrix DMET 1.0平台测试170种药物处置基因中的1256种遗传变异。分析遗传多态性与临床反应和毒性的关系。三个基因中的10个snp可能与治疗反应相关：PPARdelta、SULT1C2和CHST3。8个基因中的11个snp与治疗毒性相关：SPG7、CHST3、CYP2D6、NAT2、ABCC6、ATP7A、CYP4B1和SLC10A2。DMET基因分型结果与直接测序结果的一致性大于96%。这些发现强调了非cyp450代谢酶和转运蛋白可能在多西他赛和沙利度胺的药理学中发挥的作用。VEGFR2多态性与贝伐单抗/索拉非尼：高血压（HT）和手足皮肤反应（HFSR）可能与贝伐单抗和索拉非尼的活性有关。我们假设这些毒性与这些药物的有利结果相对应，HT和HFSR是一致的，VEGFR2基因型变异与毒性和临床结果相关。比较毒性发生率、VEGFR2 H472Q和V297I状态与临床结果。在使用贝伐单抗治疗前列腺癌患者（31.5个月vs 14.9个月，n=60， P=0.0009）和贝伐单抗和索拉非尼治疗实体肿瘤患者（11.9个月vs 3.7个月，n=27， P=0.052）的试验中，接受贝伐单抗治疗的患者在接受贝伐单抗治疗后的PFS比没有这种毒性的患者更长。在索拉非尼和贝伐单抗联合治疗后，HT也与5倍的OS获益相关（5.7对29.0个月，P=0.0068）。HFSR是索拉非尼治疗期间PFS延长的标志（6.1 vs 3.7个月，n=113， P=0.0003）。在接受贝伐单抗和/或索拉非尼治疗的患者中，高温疗法是HFSR的一个危险因素。VEGFR2 H472Q变异等位基因的携带者发生HT和HFSR的风险更高。本研究表明，HT和HFSR可能是有利临床结果的标志，HT的发展可能是HFSR的标志，VEGFR2等位基因可能与贝伐单抗和/或索拉非尼治疗期间毒性的发展有关。XRCC1多态性与放疗：XRCC1基因多态性与前列腺癌放射治疗相关：放射治疗是局限性前列腺癌的一种潜在的、重要的治疗选择。然而，在放射治疗后8年，即使在风险最高的患者亚群中，大约10%的患者会经历临床疾病复发。在此，我们研究了DNA修复的五个分子标记。513例CRPC患者，其中放疗患者284例，未放疗患者229例，健康个体152例，对DNA切除修复基因ERCC1 N118N （500C&gt；T）、XPD K751Q （2282A&gt；C）、XRCC1 R194W （685C&gt；T）、XRCC1 R399Q （1301G&gt；A）、PARP1 V762A (2446T&gt；C) 5种多态性进行基因分型。评估的多态性没有显示出患者组和健康对照组之间的差异，也没有显示出与生存相关的趋势。然而，在放射治疗亚组中，中位生存时间与XRCC1单倍型相关。R399Q AA/R194W CC单倍型患者中位生存时间为11.75年，R399Q AG/R194W CC单倍型患者中位生存时间为12.17年，R399Q AG/R194W CT单倍型患者中位生存时间为66.65年，R399Q GG/R194W CT单倍型患者中位生存时间为6.21年（p = 0.034）。当对所有患者进行调查时，并没有发现这种关联。我们得出结论，XRCC1基因多态性可能影响局部前列腺癌放疗患者的预后。ERCC1多态性与铂基化疗：ERCC1的遗传多态性被认为有助于改变对铂基化疗的敏感性。虽然ERCC1 N118N （500 C&gt；T, rs11615）是研究最多的多态性，但这种多态性对铂基化疗的影响尚不清楚。本研究首次探讨了ERCC1 N118N对基因表达和铂敏感性的功能影响。本研究的目的是探讨在一个控制良好的生物系统中，含有沉默突变变异等位基因的AAT的密码子使用频率降低是否对ERCC1有功能影响。具体来说，将带有C或T等位基因的ERCC1 cDNA克隆导入ERCC1缺陷细胞系UV20，并检测这两个等位基因对ERCC1转录、翻译和铂敏感性的影响。ERCC1 mRNA和蛋白表达水平在顺铂治疗后均升高，在治疗后4h达到峰值，但两个等位基因之间无差异（p>0.05）。尽管转染野生型和多态等位基因的细胞之间没有差异，但在铂暴露后，携带ERCC1的细胞的存活率明显高于亲本细胞系（p<0.0001）。这些数据表明，N118N本身与ERCC1表达或功能的表型差异无关，而这种多态性可能与其他致病变异或单倍型有关。

Our laboratory has a strong interest in pharmacogenetics. We have been active in studying how germline genetic variants can alter pharmacokinetics, response, and toxicity of various anticancer agents, thereby contributing to inter-individual variation in clinical outcomes in therapies with an already narrow therapeutic window. We have established a molecular link between these polymorphisms and their phenotype as it relates to drug treatment. Most of our work has been focused on genetic variations in drug metabolism and transporting candidate genes such as ABCB1 (P-glycoprotein, MDR1), ABCG2 (BCRP), SLCO1B3 (OATP1B3, OATP8), CYP3A4, CYP3A5, CYP1B1, CYP2C19, CYP2D6, UGT1A1, UGT1A9 and several others. We are also interested in non-candidate gene approaches where large numbers of polymorphisms are explored to establish a relationship with clinical outcome, and experiments are conducted to validate potential causative alleles resulting from exploratory scanning. We have worked with Affymetrix to beta-test the DMET chip that contains 1,256 genetic variations in 170 drug disposition genes, and are currently establishing a clinical trial where patients treated at the NCI will be genotyped with the DMET chip to explore potential links between these genes and various treatments of several cancers. We are currently making progress in validating the results from the initial DMET chip experiments. While many of these studies have been conducted in order to explain some of the genetic influence on pharmacokinetic variability, we also have a strong interest in clarifying genetic markers of pharmacodynamics and therapeutic outcome of several major anticancer agents since this field has been rather poorly studied. DMET: Docetaxel exhibits significant inter-individual variation in its pharmacokinetic and toxicity profile. Thalidomide is an active anticancer agent and also exhibits wide pharmacologic variation. Past pharmacogenetic research has not explained this variation. Patients with prostate cancer enrolled in a randomized phase II trial using docetaxel and thalidomide versus docetaxel alone were genotyped using the Affymetrix DMET 1.0 platform, which tests for 1,256 genetic variations in 170 drug disposition genes. Genetic polymorphisms were analyzed for associations with clinical response and toxicity. Ten SNPs in three genes were potentially associated with response to therapy: PPARdelta, SULT1C2, and CHST3. Eleven SNPs in eight genes were associated with toxicities to treatment: SPG7, CHST3, CYP2D6, NAT2, ABCC6, ATP7A, CYP4B1, and SLC10A2. Genotyping results between DMET and direct sequencing showed greater than 96% concordance. These findings highlight the role that non-CYP450 metabolizing enzymes and transporters may play in the pharmacology of docetaxel and thalidomide. VEGFR2 polymorphism & bevacizumab/sorafenib: Hypertension (HT) and hand-foot skin reactions (HFSR) may be related to the activity of bevacizumab and sorafenib. We hypothesized that these toxicities would correspond to favorable outcome in these drugs, that HT and HFSR would coincide, and that VEGFR2 genotypic variation would be related to toxicity and clinical outcomes. Toxicity incidence and VEGFR2 H472Q and V297I status were compared to clinical outcomes. Individuals experiencing HT had longer PFS following bevacizumab therapy than those without this toxicity in trials utilizing bevacizumab in patients with prostate cancer (31.5 vs 14.9mo, n=60, P=0.0009), and bevacizumab and sorafenib in patients with solid tumors (11.9 vs 3.7 mo, n=27, P=0.052). HT was also linked to a >5-fold OS benefit after sorafenib and bevacizumab cotherapy (5.7 versus 29.0 mo, P=0.0068). HFSR was a marker for prolonged PFS during sorafenib therapy (6.1 vs 3.7 mo, n=113, P=0.0003). HT was a risk factor for HFSR in patients treated with bevacizumab and/or sorafenib. Carriers of variant alleles at VEGFR2 H472Q experienced greater risk of developing HT and HFSR. This study suggests that HT and HFSR may be markers for favorable clinical outcome, HT development may be a marker for HFSR, and VEGFR2 alleles may be related to the development of toxicities during therapy with bevacizumab and/or sorafenib. XRCC1 polymorphism & radiation: Genetic polymorphisms in XRCC1 associated with radiation therapy in prostate cancer: Radiation therapy is a potentially curative, important treatment option in localized prostate cancer. However, at 8 years after radiation therapy, even in the best risk subset of patients, approximately 10% of patients will experience clinical disease recurrence. Herein, we investigated five molecular markers of DNA repair. 513 patients with castrate-resistant prostate cancer (CRPC), including 284 patients who received radiotherapy, 229 patients without radiotherapy, and 152 healthy individuals were genotyped for 5 polymorphisms in DNA excision repair genes: ERCC1 N118N (500C>T), XPD K751Q (2282A>C), XRCC1 R194W (685C>T), XRCC1 R399Q (1301G>A) and PARP1 V762A (2446T>C). The polymorphisms evaluated did not show differences between the patient group and the healthy controls, nor did they show a trend toward an association with survival. However, in the radiation treated subgroup, the median survival time was associated with the XRCC1 haplotype. The median survival time was 11.75 years for patients with the R399Q AA/R194W CC haplotype, 12.17 years for patients with the R399Q AG/R194W CC haplotype, 66.65 years for patients with the R399Q AG/R194W CT haplotype, and 6.21 years for patients with the R399Q GG/R194W CT haplotype (p = 0.034). This association was not found when all patients were investigated. We conclude that the genetic polymorphisms in XRCC1 may affect the outcome in patients who received radiotherapy for localized prostate cancer. ERCC1 polymorphism & platinum-base chemotherapy: Genetic polymorphisms in ERCC1 are thought to contribute to altered sensitivity to platinum-based chemotherapy. Although ERCC1 N118N (500 C>T, rs11615) is the most studied polymorphism, the impact of this polymorphism on platinum-based chemotherapy remains unclear. This is the first study in which the functional impact of ERCC1 N118N on gene expression and platinum sensitivity was explored. The aim of this study is to investigate if the reduced codon usage frequency of AAT, which contains the variant allele of the silent mutation, has functional impact on ERCC1 in a well-controlled biological system. Specifically, the ERCC1 cDNA clone with either the C or T allele was introduced into an ERCC1 deficient cell line, UV20, and assayed for the effect of the two alleles on ERCC1 transcription, translation and platinum sensitivity. Both ERCC1 mRNA and protein expression levels increased upon cisplatin treatment, peaking at 4h post-treatment, however there were no differences between the two alleles (p>0.05). Cells complemented with ERCC1 showed significantly higher survival proportion than the parental cell line following platinum exposure (p<0.0001), although no differences were observed between the cells transfected with the wild type or the polymorphic allele. These data suggest that N118N itself is not related to the phenotypic differences in ERCC1 expression or function, but rather this polymorphism may be linked to other causative variants or haplotypes.