C. Xu

Pazopanib Efficacy in Renal Cell Carcinoma: Evidence for Predictive Genetic Markers in Angiogenesis-Related and Exposure-Related Genes

PURPOSEnPazopanib, an oral angiogenesis inhibitor, is approved for the treatment of advanced renal cell carcinoma (RCC). Response to pazopanib monotherapy varies between patients, and no validated biomarkers predictive of treatment outcome have been identified. We tested the hypothesis that this variability is partially dependent on germline genetic variants that may affect pazopanib exposure or angiogenesis pathways.nnnPATIENTS AND METHODSnTwenty-seven functional polymorphisms within 13 genes were evaluated in 397 patients with RCC. Genetic association with progression-free survival (PFS) and objective response rate (RR) was analyzed using the Cox proportional hazards model and proportional odds model, respectively.nnnRESULTSnThree polymorphisms in IL8 and HIF1A and five polymorphisms in HIF1A, NR1I2, and VEGFA showed nominally significant association (P ≤ .05) with PFS and RR, respectively. Compared with the wild-type AA genotype (median PFS, 48 weeks), the IL8 2767TT variant genotype showed inferior PFS (27 weeks, P = .009). The HIF1A 1790AG genotype was associated with inferior PFS and reduced RR, compared with the wild-type GG genotype (median PFS, 20 v 44 weeks; P = .03; RR, 30% v 43%, P = .02). Reductions in RR were detected for the NR1I2 -25385TT genotype, compared with the wild-type CC genotype (37% v 50%, P = .03), and for the VEGFA -1498CC genotype compared with the TT genotypes (33% v 51%).nnnCONCLUSIONnGermline variants in angiogenesis- and exposure-related genes may predict treatment response to pazopanib monotherapy in patients with RCC. If validated, these markers may explain why certain patients fail antiangiogenesis therapy and they may support the use of alternative strategies to circumvent this issue.

Association of the hemochromatosis gene with pazopanib-induced transaminase elevation in renal cell carcinoma

BACKGROUND & AIMSnPazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC.nnnMETHODSnData from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels.nnnRESULTSnOf 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure.nnnCONCLUSIONSnThe rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.

Linkage disequilibrium mapping identifies a 390 kb region associated with CYP2D6 poor drug metabolising activity

The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as ‘cases’. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 × 10−4 to 4.54 × 10−35). Haplotype analysis revealed more significant levels of association (P = 3.54 × 10−56). Strong (D′ > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.

Genetic characterization to improve interpretation and clinical management of hepatotoxicity caused by tyrosine kinase inhibitors

Tyrosine kinase inhibitors (TKIs) represent important therapeutic alternatives to, or combinations with, traditional cytotoxic chemotherapy. Despite their selective molecular targeting and demonstrated clinical benefit, TKIs produce a range of serious adverse events, including drug-induced liver injury, that require careful patient management to maintain treatment benefit without harm. Genetic characterization of serious adverse events can identify mechanisms of injury and improve safety risk management. This review presents pharmacogenetic comparisons of two approved TKIs, lapatinib and pazopanib, which reveal different mechanisms of injury and inform the characteristics and risk of serious liver injury in treated patients. The data presented demonstrate the utility of genetic studies to investigate drug-induced liver injury and potentially support its management in patients.

Concomitant use of pazopanib and simvastatin increases the risk of transaminase elevations in patients with cancer

Alanine aminotransferase (ALT) elevations were reported in oncology trials of the oral angiogenesis inhibitor pazopanib [1]. Statins, which are widely used to treat hypercholesterolemia [2], are also associated with ALT elevations. As pazopanib and statins are substrates for the same key metabolizing enzymes [e.g. cytochrome P450 3A4 (CYP3A4)] and drug transporters [e.g. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP)] [2–3], it is plausible that concomitant administration of pazopanib and statins may alter their systemic and/or hepatic exposures, leading to increased toxicities such as liver injury. Using individual patient data from 11 pazopanib clinical studies for the treatment of cancer (n = 976), we evaluated the effects of concomitant pazopanib and statin use on the incidence of ALT elevation [≥3 × upper limit of normal (ULN)]. The incidence of ALT≥ 3 × ULN was 21% in patients who received both pazopanib and any statin and 14% in patients who did not receive statins (P = 0.10; Table 1). Simvastatin and atorvastatin were the two most commonly used statins in this patient population. The incidence of ALT≥ 3 × ULN (27%) was significantly higher in simvastatin users than in patients who did not receive statins (P = 0.04; Table 1). Although the incidence of ALT≥ 3 × ULN was also higher in atorvastatin users (17%) than in patients who did not receive statins (14%), this difference did not reach statistical significance (P = 0.59). Among patients with ALT≥ 3 × ULN who received both pazopanib and simvastatin, ALT recovery to <2.5 × ULN (grade ≤1 by common toxicity criteria v3) was documented for 10/11 patients (91%) after either (i) no alteration for pazopanib and simvastatin therapy (n = 2); (ii) discontinuation of simvastatin only (n = 2); (iii) discontinuation of pazopanib only (n = 4); or (iv) discontinuation of both simvastatin and pazopanib (n = 2). There were insufficient follow-up ALT data to assess recovery for the one remaining patient after pazopanib was discontinued. The ALT recovery rate (to <2.5 × ULN) for patients receiving concomitant pazopanib and simvastatin was comparable with that seen in the larger group of pazopanib-treated study patients with renal cell carcinoma who had ALT≥ 3 × ULN (96/106, 91%) [4]. Exploratory pharmacogenetic analysis revealed that the ABCG2 (BCRP) 421C>A polymorphism may be associated with ALT elevation in patients taking pazopanib and simvastatin. Those with the variant 421C>A allele had a higher incidence of ALT≥ 3 × ULN (5/7, 71%) compared with patients with wild-type genotype (2/21, 10%; odds ratio = 19.6, 95% confidence interval 1.9–231.6; P = 0.004). This polymorphism was not associated with ALT elevations in pazopanib-treated patients without concurrent use of statins. This study showed that concomitant use of pazopanib and simvastatin increased the risk of transaminase elevations in patients with cancer. In addition to implementing the recommended dose modification guidelines for pazopanib, discontinuation of simvastatin should be considered to manage the risk of liver injury in cancer patients receiving both medications. Furthermore, hepatotoxicity/transaminase elevations have been reported in patients receiving other multi-tyrosine kinase inhibitors (e.g. imatinib, erlotinib, lapatinib, nilotinib, and sunitinib) that are also metabolized primarily by CYP3A4 [5]. The effect of concomitant administration of these tyrosine kinase inhibitors and statins on potential hepatotoxicity remains to be determined.

Identification of a Variant in KDR Associated with Serum VEGFR2 and Pharmacodynamics of Pazopanib

Purpose: VEGF receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular VEGFR2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors. Experimental Design: We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 patients with advanced solid tumor with baseline [sVEGFR2] measurements, and in 121 patients with renal carcinoma with [sVEGFR2] measured before and during pazopanib therapy. Results: rs34231037 (C482R) in KDR, the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance (P = 2.7 × 10−37). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size (P = 0.025). Furthermore, rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib (P = 0.01). Conclusion: Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germline variants in KDR may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of VEGFR2 kinase inhibitors. Clin Cancer Res; 21(2); 365–72. ©2014 AACR.

Hyperbilirubinemia in pazopanib- or sunitinib-treated patients in COMPARZ is associated with UGT1A1 polymorphisms

Pazopanib and sunitinib are angiogenesis inhibitors approved for treatment of advanced renal cell carcinoma. Treatmentassociated elevations in alanine aminotransferase (ALT) and bilirubin have been reported for both therapies [1]. UGT1A1 polymorphisms are known to cause reduced expression/ activity of UGT1A1 and predispose individuals to Gilbert’s syndrome, a benign form of episodic jaundice [2, 3]. Our analyses of previous pazopanib clinical trials found that UGT1A1 *28 was associated with on-treatment hyperbilirubinemia [4]. This study extended our previous analysis of UGT1A1*28 to additional alleles (*36, *37, and *6) and investigated their association with on-therapy serum total bilirubin in COMPARZ (NCT00720941 and NCT01147822), a phase III, randomized, clinical trial comparing pazopanib versus sunitinib for the treatment of advanced renal cell carcinoma [1]. Of 1110 randomized participants, on-therapy hyperbilirubinemia [total bilirubin ≥1.5 × upper limit of the normal range (ULN)] was observed in 16% (89/557) of pazopanib-arm patients and 9% (49/553) of sunitinib-arm patients. In the pharmacogenetic population (patients who received at least one dose of study treatment, consented, and were successfully genotyped, n = 719), the incidence of hyperbilirubinemia was 17% (62 of 369) for pazopanib and 10% (34 of 350) for sunitinib. The incidence of hyperbilirubinemia varies by UGT1A1 genotypes for both therapies (Figure 1). Logistic regression adjusted for ancestry principal components was used to compare hyperbilirubinemia cases (pazopanib n = 62; sunitinib n = 34) against controls (pazopanib n = 211; sunitinib n = 212). Controls were defined as patients with all on-therapy bilirubin ≤1 × ULN and treatment exposure equal to or greater than median exposure until bilirubin elevation in cases (defined in the supplementary materials, available at Annals of Oncology online). Patients with predicted reduced UGT1A1 function (homozygous or inferred compound heterozygous for *28, *37, and *6) had higher baseline bilirubin and were more likely to experience hyperbilirubinemia when receiving either pazopanib (P = 7.7 × 10) or sunitinib (P = 1.7 × 10), with odds ratios (95% confidence interval) 9.97 (4.13–24.03) and 5.83 (2.04–16.68), respectively. After adjusting for baseline bilirubin, patients with predicted, reduced UGT1A1 function remained more likely to experience hyperbilirubinemia when receiving pazopanib (P = 0.012) or sunitinib (P = 0.024), with odds ratios (95% confidence interval) 3.65 (1.31–10.16) and 4.51 (1.26–16.11), respectively. UGT1A1 genotypes were not significantly associated with ALT ≥3 × ULN in pazopanib-treated patients. A borderline significant association (P = 0.030) was seen between UGT1A1 genotypes with reduced function (predicted) and decreased incidence of ALT ≥3 × ULN in sunitinib-treated patients; additional studies are required to confirm this observation. Pazopanib is a potent inhibitor of UGT1A1 in vitro [4]. Our data suggest that hyperbilirubinemia in pazopanibtreated patients may be the result of inhibition of UGT1A1 combined with effects of genetic variants in the UGT1A1 gene. We expect this would result in higher levels of unconjugated hyperbilirubinemia, usually associated with a benign clinical course. Sunitinib, however, does not inhibit UGT1A1 [5]. To our knowledge, this is the first study reporting the association between UGT1A1 genotype and hyperbilirubinemia in patients receiving sunitinib. Our data suggest that some instances of hyperbilirubinemia in patients treated with pazopanib or sunitinib may be benign manifestations of Gilbert’s syndrome. Bilirubin fractionation or, if not available, UGT1A1 genotyping, would enable further characterization of liver safety risk and help in making treatment decisions.

Different effects of the BIM deletion polymorphism on treatment of solid tumors by the tyrosine kinase inhibitors (TKI) pazopanib, sunitinib, and lapatinib

FISH ‘borderline’ tumors (i.e. 15%–20% of rearranged nuclei) emphasizing potential issues in ALK FISH analysis [2]. They especially reported the particular cases of two FISH− IHC+ tumors with ALK amplification (i.e. high copy number corresponding to more than six copies per nucleus). Within 26 ALK FISH+ tumors (i.e. with more than 15% of rearranged FISH nuclei using the FDA-approved ALK break-apart FISH probe), no tumor presented ALK amplification. ALK copy number gains have been described in NSCLC. Although ALK amplification in ALK-rearranged tumors seems to be rare, few studies have been conducted in these tumors [3– 5]. We report here a case of ALK-rearranged and amplified tumor that was ALK IHC negative. We identified a single case of both ALK-rearranged and amplified tumor among ∼70 cases of ALK FISH positive NSCLC (∼7% of the NSCLC samples analyzed in our institutional cancer molecular genetics platform). This represents <2% of ALK-rearranged NSCLC and 0.1% of unselected NSCLC in our institution. The sample was a metastasis from the abdominal wall in a patient with a voluminous lung tumor. The ALK rearrangement consisted in both FISH split and single 30 signals in 30% of tumor cells (mean 2.8 split and 1.5 single 30 signals per nucleus). There was a mean number of 4.5 fused signals per nucleus. Adding spit and single 30 signals resulted in a mean number of 8.8 ALK copies per nucleus whereas the mean number of chromosome 2 per nucleus was 3.5 with an ALK/chromosome 2 ratio of 2.5. This was consistent with an ALK amplification. The ALK IHC analysis (clone 5A4, 1:25, Clinisciences) was negative (score 0) (Figure 1). To date, this 46-year-old smoker woman, with KRAS, EGFR and BRAF nonmutated status, has not been treated by crizotinib. This particular case illustrates some challenges in ALK rearrangement screening. First, ALK amplification, with FISH clusters in some cells as encountered in our case can impair the detection of positive signals within tumor cells and in this manner requires careful examination of these cases. Second, if ALK copy number gains are frequent in ALK nonrearranged tumors, ALK-amplified tumors are rare in ALK-rearranged tumors, as previously described [1, 3–5]. Finally, as previously mentioned in some studies, we agree with Ilie et al. and think that both IHC and FISH analyses should be carried out to avoid ALK status misdiagnosis [1, 2]. We should be aware of these particular cases because this rare ALK double alteration would have been missed by an IHC-based prescreening, as proposed in some studies.

Genetic evaluation of fatigue in pazopanib-treated patients (pts) with advanced renal cell carcinoma (RCC).

e15072 Background: Fatigue is a common adverse effect of many cancer drugs, which can be debilitating for pts and have negative implications on their physical, mental, and social well-being. Pazopanib is an oral angiogenesis inhibitor indicated for the treatment of advanced RCC. Pts treated with pazopanib experience fatigue to varying degrees. This study sought to explore genetic factors which may contribute to treatment related fatigue in RCC.nnnMETHODSnDNA was collected from consenting pts with advanced RCC for genetic study from three pazopanib clinical studies; VEG102616 (ph II), VEG105192 (ph III), and VEG107769 (open label extension study of VEG105192). Pts were defined as fatigue cases (NCI-CTCv3 grade ≥ 2) or fatigue controls (no event). A total of 1999 SNPs in 58 candidate genes were evaluated. These genes were selected for a role in the pharmacokinetic and pharmacodynamic pathway of pazopanib or for a suggestive association with fatigue in the literature. Logistic regression was used to evaluate the effect of the genotypes on the case-control status of fatigue.nnnRESULTSnNone of the SNPs were associated with fatigue at the multiple-testing corrected significance level (p<6 x 10-6). However, 11 SNPs in four cytokine-encoding genes (IL1A, IL1RN, IL17RA, and IL8RA) met pre-specified criteria for a suggestive association with fatigue (p<0.01). The minor alleles in IL1A, IL17RA, or IL8RA were more often observed in individuals without fatigue event, suggesting that these alleles may confer a protective effect. Conversely, IL1RN minor alleles were observed at a higher frequency in individuals with moderate to severe fatigue (grade ≥ 2), and therefore pts carrying these alleles may be at a higher risk of developing fatigue with pazopanib treatment. IL1RN rs315948 genotypes also showed a suggestion association with fatigue severity.nnnCONCLUSIONSnCandidate gene analyses revealed that SNPs in cytokine-encoding genes may be associated with pazopanib related fatigue. However, as none of the SNPs passed the significance level of multiple testing corrections, further evaluation in independent data sets is required to confirm or refute these findings.