The burden of rare variants in DPYS gene is a novel predictor of the risk of developing severe fluoropyrimidine-related toxicity.

BACKGROUND: Despite a growing number of publications highlighting the potential impact on the therapy outcome, rare genetic variants (minor allele frequency < 1%) in genes associated to drug adsorption, distribution, metabolism, and elimination are poorly studied. Previously, rare germline DPYD missense variants were shown to identify a subset of fluoropyrimidine-treated patients at high risk for severe toxicity. Here, we investigate the impact of rare genetic variants in a panel of 54 other fluoropyrimidine-related genes on the risk of severe toxicity. METHODS: The coding sequence and untranslated regions of 54 genes related to fluoropyrimidine pharmacokinetics/pharmacodynamics were analyzed by next-generation sequencing in 120 patients developing grade 3-5 toxicity (NCI-CTC vs3.0) and 104 matched controls. Sequence Kernel Association Test (SKAT) analysis was used to select genes with a burden of genetic variants significantly associated with risk of severe toxicity. The statistical association of common and rare genetic variants in selected genes was further investigated. The functional impact of genetic variants was assessed using two different in silico prediction tools (Predict2SNP; ADME Prediction Framework). RESULTS: SKAT analysis highlighted DPYS and PPARD as genes with a genetic mutational burden significantly associated with risk of severe fluoropyrimidine-related toxicity (Bonferroni adjusted P = 0.024 and P = 0.039, respectively). Looking more closely at allele frequency, the burden of rare DPYS variants was significantly higher in patients with toxicity compared with controls (P = 0.047, Mann-Whitney test). Carrying at least one rare DPYS variant was associated with an approximately fourfold higher risk of severe cumulative (OR = 4.08, P = 0.030) and acute (OR = 4.21, P = 0.082) toxicity. The burden of PPARD rare genetic variants was not significantly related to toxicity. Some common variants with predictive value in DPYS and PPARD were also identified: DPYS rs143004875-T and PPARD rs2016520-T variants predicted an increased risk of severe cumulative (P = 0.002 and P = 0.001, respectively) and acute (P = 0.005 and P = 0.0001, respectively) toxicity. CONCLUSION: This work demonstrated that the rare mutational burden of DPYS, a gene strictly cooperating with DPYD in the catabolic pathway of fluoropyrimidines, is a promising pharmacogenetic marker for precision dosing of fluoropyrimidines. Additionally, some common genetic polymorphisms in DPYS and PPARD were identified as promising predictive markers that warrant further investigation.

Implementation of pre-emptive testing of a pharmacogenomic panel in clinical practice: Where do we stand?

Adverse drug reactions (ADRs) account for a large proportion of hospitalizations among adults and are more common in multimorbid patients, worsening clinical outcomes and burdening healthcare resources. Over the past decade, pharmacogenomics has been developed as a practical tool for optimizing treatment outcomes by mitigating the risk of ADRs. Some single-gene reactive tests are already used in clinical practice, including the DPYD test for fluoropyrimidines, which demonstrates how integrating pharmacogenomic data into routine care can improve patient safety in a cost-effective manner. The evolution from reactive single-gene testing to comprehensive pre-emptive genotyping panels holds great potential for refining drug prescribing practices. Several implementation projects have been conducted to test the feasibility of applying different genetic panels in clinical practice. Recently, the results of a large prospective randomized trial in Europe (the PREPARE study by Ubiquitous Pharmacogenomics consortium) have provided the first evidence that prospective application of a pre-emptive pharmacogenomic test panel in clinical practice, in seven European healthcare systems, is feasible and yielded a 30% reduction in the risk of developing clinically relevant toxicities. Nevertheless, some important questions remain unanswered and will hopefully be addressed by future dedicated studies. These issues include the cost-effectiveness of applying a pre-emptive genotyping panel, the role of multiple co-medications, the transferability of currently tested pharmacogenetic guidelines among patients of non-European origin and the impact of rare pharmacogenetic variants that are not detected by currently used genotyping approaches.

CYP2D6 and CYP2C8 pharmacogenetics and pharmacological interactions to predict imatinib plasmatic exposure in GIST patients.

AIMS: Patients on treatment with oral fixed dose imatinib are frequently under- or overexposed to the drug. We investigated the association between the gene activity score (GAS) of imatinib-metabolizing cytochromes (CYP3A4, CYP3A5, CYP2D6, CYP2C9, CYP2C19, CYP2C8) and imatinib and nor-imatinib exposure. We also investigated the impact of concurrent drug-drug-interactions (DDIs) on the association between GAS and imatinib exposure. METHODS: Serial plasma samples were collected from 33 GIST patients treated with imatinib 400 mg daily within a prospective clinical trial. Imatinib and nor-imatinib Ctrough were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Genetic polymorphisms with a functional impact on imatinib-metabolizing cytochromes were identified and a GAS was calculated for each gene. A DDI-adjusted GAS was also generated. RESULTS: Imatinib and nor-imatinib Ctrough were measured in 161 plasma samples. CYP2D6 GAS and metabolizer status based on genotype were associated with imatinib and (imatinib + nor-imatinib) Ctrough . CYP2D6 poor and intermediate metabolizers were predicted to have a lower nor-imatinib/imatinib metabolic ratio than normal metabolizers (0.197 and 0.193 vs. 0.247, P = .0205), whereas CYP2C8*3 carriers had a higher ratio than CYP2C8*1/*1 patients (0.263 vs. 0.201, P = .0220). CYP2C9 metabolizer status was inversely related to the metabolic ratio with an effect probably driven by the linkage disequilibrium between CYP2C9*2 and CYP2C8*3. The CYP2D6 DDI-adjusted GAS was still predictive of imatinib exposure. CONCLUSIONS: These findings highlight that CYP2D6 plays a major role in imatinib pharmacokinetics, but other players (i.e., CYP2C8) may influence imatinib exposure. These findings could drive the selection of patients more susceptible to imatinib under- or overexposure who could be candidates for personalized treatment and intensified monitoring strategies.

Impact of ABCG2 and ABCB1 Polymorphisms on Imatinib Plasmatic Exposure: An Original Work and Meta-Analysis.

Adequate imatinib plasma levels are necessary to guarantee an efficacious and safe treatment in gastrointestinal stromal tumor (GIST) and chronic myeloid leukemia (CML) patients. Imatinib is a substrate of the drug transporters ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) that can affect its plasma concentration. In the present study, the association between three genetic polymorphisms in ABCB1 (rs1045642, rs2032582, rs1128503) and one in ABCG2 (rs2231142) and the imatinib plasma trough concentration (Ctrough) was investigated in 33 GIST patients enrolled in a prospective clinical trial. The results of the study were meta-analyzed with those of other seven studies (including a total of 649 patients) selected from the literature through a systematic review process. The ABCG2 c.421C>A genotype demonstrated, in our cohort of patients, a borderline association with imatinib plasma trough levels that became significant in the meta-analysis. Specifically, homozygous carriers of the ABCG2 c.421 A allele showed higher imatinib plasma Ctrough with respect to the CC/CA carriers (Ctrough, 1463.2 ng/mL AA, vs. 1196.6 ng/mL CC + AC, p = 0.04) in 293 patients eligible for the evaluation of this polymorphism in the meta-analysis. The results remained significant under the additive model. No significant association could be described between ABCB1 polymorphisms and imatinib Ctrough, neither in our cohort nor in the meta-analysis. In conclusion, our results and the available literature studies sustain an association between ABCG2 c.421C>A and imatinib plasma Ctrough in GIST and CML patients.

New insights into the pharmacological, immunological, and CAR-T-cell approaches in the treatment of hepatocellular carcinoma.

The tyrosine kinase inhibitor (TKI) sorafenib continues to be the anchor drug in the treatment of advanced stage hepatocellular carcinoma (HCC). Other TKIs as well as immune checkpoint inhibitors (ICIs) have also been approved, however the response rates remain poor and heterogeneous among HCC patients, largely due to antitumor drug resistance. Studies aimed at identifying novel biomarkers and developing new strategies to improve the response to current treatment and to overcome drug resistance, are urgently needed. Germline or somatic mutations, neoantigens, and an immunotolerogenic state against constant inflammatory stimuli in the liver, are crucial for the anti-tumor response. A pharmacogenetic approach has been attempted considering germline polymorphisms in genes encoding for proteins involved in drug-targeted pathways. Single gene and comprehensive multi-gene somatic profiling approaches have been adopted in HCC to identify tumor sensitivity scores and immunogenic profiles that can be exploited for new biomarkers and innovative therapeutic approaches. However, the high genomic heterogeneity of tumors and lack of molecularly targeted agents, hamper the discovery of specific molecular markers of resistance to therapy. Adoptive cell therapy with chimeric antigen receptor redirected T (CAR-T) cells targeting specific tumor-associated antigens (TAAs) was proposed recently. The specificity of the chosen TAA, an efficient homing of CAR-T cells to the tumor site, and the ability of CAR-T cells to survive in the tumor microenvironment are central factors in the success of CAR-T therapy. The current review describes the principal systemic treatments for HCC and the molecular evidence regarding potential predictive host and somatic genetic markers, as well as the emerging strategy of liquid biopsy for disease monitoring. Novel immunotherapeutic approaches for HCC treatment, including the use of ICIs and CAR-T, as well as strategies to overcome drug resistance, are discussed.

A TGF-ß associated genetic score to define prognosis and platinum sensitivity in advanced epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (EOC) is usually diagnosed at advanced stages with highly variable clinical outcomes, even among patients with similar clinical characteristics and treatments. Host immune system plays a pivotal role in EOC pathogenesis and progression. Here, we assessed the clinical significance of 192 single nucleotide polymorphisms (SNPs) on 34 immune-system related genes in EOC patients. METHODS: Two hundred and thirty advanced EOC patients treated with platinum-based chemotherapy were included. Germ-line DNA was analyzed with Illumina GoldenGate Genotyping Assay. RESULTS: Nineteen polymorphisms were significantly associated with overall survival (OS), 17 with progression free survival (PFS) and 20 with platinum-free interval (PFI). Of the 8 polymorphisms associated with all three outcomes, 7 SNPs belonged to genes involved in the TGF-ß pathway. A genetic score was built considering the unfavourable genotypes (UGs) of these 7 polymorphisms (group 0-2 UGs: presence of 0, 1, or 2 UGs; group 3-4 UGs: 3 or 4 UGs; group 5-7: 5, 6, or 7 UGs). According to this score, OS decreased as the number of UGs increased (median OS: 0-2 UGs = not reached, 3-4 UGs = 44.6 and 5-7 UGs = 19.3 months, p < 0.0001). The same trend was observed also for PFS (median PFS: 0-2 UGs = 21.5, 3-4 UGs = 17.3 and 5-7 UGs = 11 months, p < 0.0001) and PFI (median PFI: 0-2 UGs = 16.6, 3-4 UGs = 9.8 and 5-7 UGs = 3.8 months, p < 0.0001). The score was validated by permutation analysis. CONCLUSIONS: The proposed TGF-ß pathway score could be useful to define prognosis and platinum sensitivity of advanced EOC patients.

Fluorinated steroids are not superior to any treatment to ameliorate the outcome of autoimmune mediated congenital heart block: a systematic review of the literature and meta-analysis.

OBJECTIVES: Fluorinated steroids are largely the therapeutic approach of autoimmune mediated congenital heart block (CHB). We performed a meta-analysis to assess the efficacy of fluorinated steroids for the treatment of CHB. METHODS: Studies evaluating the efficacy of fluorinated steroids versus no treatment in CHB patients were identified in electronic databases. Random-effects model was used to pool odds ratio (OR) (with 95% CI) of live births as the primary outcome. ORs of CHB progression, pacemaker implantation and extranodal disease were the secondary outcome. Subgroup analysis according to CHB grade and study type was performed. RESULTS: Data from nine studies involving 747 patients were analysed. The overall live birth rates were 86.8% and 86.7%, respectively, in the fluorinated steroids exposed foetuses and in the non-exposed ones. Fluorinated steroids did not ameliorate overall survival in CHB (OR 1.02; 95% CI: 0.65-1.61) with any significant statistical heterogeneity between studies (I2 0%, p=0.45). No significant differences for the progression of CHB, the pacing and the presence of extranodal disease were observed. Subgroup analysis revealed a significant protective role of fluorinated steroids for survival in 3rd degree CHB and for pacing in monocentric studies, OR 4.07; 95% CI: 1.10-15.08 and OR 0.15; 95% CI: 0.02-0.99, respectively. CONCLUSIONS: This meta-analysis shows that fluorinated steroids are not superior to any treatment in patients with CHB in terms of live birth, prevention of progression of incomplete CHB, pacemaker implantation and extranodal disease. Thus, considering their side effects, their use in CHB patients should be discouraged.

CDK4/6 Inhibitors in Breast Cancer Treatment: Potential Interactions with Drug, Gene, and Pathophysiological Conditions.

Palbociclib, ribociclib, and abemaciclib belong to the third generation of cyclin-dependent kinases inhibitors (CDKis), an established therapeutic class for advanced and metastatic breast cancer. Interindividual variability in the therapeutic response of CDKis has been reported and some individuals may experience increased and unexpected toxicity. This narrative review aims at identifying the factors potentially concurring at this variability for driving the most appropriate and tailored use of CDKis in the clinic. Specifically, concomitant medications, pharmacogenetic profile, and pathophysiological conditions could influence absorption, distribution, metabolism, and elimination pharmacokinetics. A personalized therapeutic approach taking into consideration all factors potentially contributing to an altered pharmacokinetic/pharmacodynamic profile could better drive safe and effective clinical use.

Host genetic profiling to increase drug safety in colorectal cancer from discovery to implementation.

Adverse events affect the pharmacological treatment of approximately 90% of colorectal cancer (CRC) patients at any stage of the disease. Chemotherapy including fluoropyrimidines, irinotecan, and oxaliplatin is the cornerstone of the pharmacological treatment of CRC. The introduction of novel targeted agents, as anti-EGFR (i.e. cetuximab, panitumumab) and antiangiogenic (i.e. bevacizumab, ziv-aflibercept, regorafenib, and ramucirumab) molecules, into the oncologist's toolbox has led to significant improvements in the life expectancy of advanced CRC patients, but with a substantial increase in toxicity burden. In this respect, pharmacogenomics has largely been applied to the personalization of CRC chemotherapy, focusing mainly on the study of inhered polymorphisms in genes encoding phase I and II enzymes, ATP-binding cassette (ABC)/solute carrier (SLC) membrane transporters, proteins involved in DNA repair, folate pathway and immune response. These research efforts have led to the identification of some validated genetic markers of chemotherapy toxicity, for fluoropyrimidines and irinotecan. No validated genetic determinants of oxaliplatin-specific toxicity, as peripheral neuropathy, has thus far been established. The contribution of host genetic markers in predicting the toxicity associated with novel targeted agents' administration is still controversial due to the heterogeneity of published data. Pharmacogenomics guidelines have been published by some international scientific consortia such as the Clinical Pharmacogenomics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG) strongly suggesting a pre-treatment dose adjustment of irinotecan based on UGT1A1*28 genotype and of fluoropyrimidines based on some DPYD genetic variants, to increase treatment safety. However, these recommendations are still poorly applied at the patient's bedside. Several ongoing projects in the U.S. and Europe are currently evaluating how pharmacogenomics can be implemented successfully in daily clinical practice. The majority of drug-related adverse events are still unexplained, and a great deal of ongoing research is aimed at improving knowledge of the role of pharmacogenomics in increasing treatment safety. In this review, the issue of pre-treatment identification of CRC patients at risk of toxicity via the analysis of patients' genetic profiles is addressed. Available pharmacogenomics guidelines with ongoing efforts to implement them in clinical practice and new exploratory markers for clinical validation are described.

Circulating-Free DNA Analysis in Hepatocellular Carcinoma: A Promising Strategy to Improve Patients' Management and Therapy Outcomes.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, representing the third leading cause of cancer-related deaths. HCC genetic characterization at the tumor level has been recently completed, highlighting how a number of genes are frequently mutated in this pathology. Actionable somatic mutations found in a HCC tumor may represent targets for innovative drugs as well as prognostic/predictive markers. Nonetheless, surgical or bioptic tissue is hardly accessible in HCC and a single tumor sample is poorly representative of the tumor genetic heterogeneity. In this context, analyzing the circulating cell-free DNA (ccfDNA) and its tumor-derived fraction (ctDNA) could represent a promising strategy of liquid biopsy. Recent data suggested that the fluctuation of the ccfDNA quantity in the plasma of HCC patients could anticipate the detection of tumor progression. The presence of somatic mutations in p53 signaling, Wnt/ß-catenin, chromatin remodeling, response to oxidative stress and telomerase maintenance pathways can also be studied in ccfDNA bypassing the need to perform a tumor biopsy. The profiling of ccfDNA fragmentation and the methylation pattern could further improve the clinical management of HCC patients. Performing a dynamic monitoring in the course of systemic treatment with sorafenib or regorafenib is a possible way to provide insights into the resistance mechanism, and to identify predictive and prognostic genetic alterations, helping the clinicians in terms of treatment decision making. This review will discuss the most recent literature data about the use of ccfDNA to monitor and improve the treatment of HCC.