The Pharmacogenetic Background of the Host Is An Independent Predictor of Outcome and Toxicity in Diffuse Large B Cell Lymphoma Treated with R-CHOP21

Scant information is available on the impact of the host genetic background as a predictor of outcome and toxicity in diffuse large B cell lymphoma (DLBCL) treated with R-CHOP. We verified whether relevant single nucleotide polymorphisms (SNPs) of the host may further refine prognostic stratification and toxicity prediction in DLBCL treated with R-CHOP21. The study was based on 106 consecutive DLBCL provided with a prospectively collected dataset. Candidate SNPs belonged to genes known to be involved in the pharmacogenetics of cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab, and included SNPs affecting:

• drug metabolism (cytochrome-P450: CYP2B6- 25504C>T, CYP2B6-15630G>T, CYP2C19-19153G>A, CYP3A4--392A>G, CYP3A4- 14365T>G, CYP3A5-6980G>A);

• drug detoxification: (gluthatione-S-transferase: GSTA1--4621T>C, GSTM1-null, GSTP1-1374A>G, GSTP1-2264C>T);

• drug transport (multidrug-resistance-related proteins: ABCC1-129623G>T, ABCC2-53394T>A, ABCC2-68692G>A, ABCB1-90855C>T, ABCB1-49691G>A, ABCG2-8824C>A, ABCG2-33G>A; and

• drug pharmacodynamics (NADPH-subunits: NCF4--368A>G, RAC2-7418T>A, CYBA-4185C>T; TP53: p53-440G>C; Fcγ receptor: FCGR2A- 4487A>G, FCGR3A-5092T>G, FCGR3A-1301T>G/A; glucocorticoid receptor: GR- 1087A>G, GR-67G>A, GR-1829C>G).

Genotyping of candidate SNPs was performed on PBMC collected at DLBCL diagnosis by SNP-minisequencing. Primary end points were event free survival (EFS) and toxicity. Associations of SNPs with primary endpoints were controlled for multiple testing by false discovery rate (FDR) analysis. Univariate log-rank analysis controlled for multiple comparisons by FDR testing identified GSTA1-- 4621C>T, CYBA-4185C>T, and ABCC2-53394T>A as predictors of EFS. GSTA1 encodes a glutathione S-transferase facilititating solubility and excretion of cyclophosphamide and its derivatives. Patients carrying the GSTA1--4621 CT/TT genotypes displayed a better EFS (3-year EFS: 68.4%; SE: 5.9%) compared to GSTA1--4621 CC carriers (3-year EFS: 39.2%; SE: 9.2%) (p=.025; q=.200). CYBA encodes a NAD(P)H oxidase subunit. Patients carrying the CYBA-4185 TT genotype displayed a poorer EFS (3-year EFS: 41.7%; SE: 12.4%) compared to CYBA-4185 CT/CC carriers (3-year EFS: 61.2%; SE: 5.8%) (p=.008; q=.156). ABCC2 encodes an ATP-binding cassette transporter (MRP2) acting as an efflux system for doxorubicin and vincristine. Patients carrying the ABCC2-53394 AT/AA genotypes displayed a poorer EFS (3-year EFS: 34.1%; SE: 11.5%) compared to ABCC2- 53394 TT carriers (3-year EFS: 64.0%; SE: 5.8%) (p=.013; q=.156). Multivariate analysis identified GSTA1--4621 CT/TT (HR: 0.39; 95% CI 0.21–0.73; p=.004) and CYBA-4185 TT (HR: 2.01; 95% CI 1.01–3.97; p=.045) as independent predictors of EFS in DLBCL treated with R-CHOP21, along with IPI 3–5 (HR: 4.88; 95% CI 2.24–8.55; p= 1.5 × 10⁻⁴). CYBA- 4185C>T and GSTA1--4621C>T also predicted outcome in DLBCL subgroups by IPI. The impact of SNPs was also evaluated for toxicity in 658 R-CHOP21 courses by logistic regression analysis adjusted for age, sex, ECOG PS, IPI, comorbidities, organ function, and doses of cyclophosphamide, doxorubicin and vincristine. Generalized estimating equations (GEE) were used to build a multivariate model for toxicity, which adjusts for the clustering of treatment courses within a patient. NCF4--368AG/GG was an independent predictor of low risk of hematologic toxicity G3-4 (HR:0.45; p=.018), infection (HR:0.46; p=.003), and cardiac toxicity (HR:0.37; p=.023). The implications of these results are twofold. First, host SNPs affecting doxorubicin and/or cyclophosphamide pharmacodynamics (CYBA-4185C>T) and detoxification (GSTA1--4621C>T) are independent predictors of outcome in DLBCL treated with R-CHOP21. Poor EFS heralded by CYBA-4185C>T, a nonsynonimous SNP of NADPH-oxidase p22phox, may be due to reduced production of reactive oxygen species (ROS) that mediate doxorubicin cytotoxicity. Favorable EFS associated with GSTA1--4621C>T may be related to reduced expression of glutathione- S-transferase A1, a phase II enzyme involved in cyclophosphamide and doxorubicin detoxification. Second, NCF4--368AG/GG, a SNP belonging to NADPH-oxidase p40phox and regulating ROS generation, has an independent protective role against both hematologic and non-hematologic toxicity.