PHARMACOGENETICS OF FLUOROPYRIMIDINES: FROM THE IMPLEMENTATION OF GUIDELINES TO IDENTIFICATION OF NOVEL GERMLINE VARIANTS RELATED TO TOXICITY

La medicina di precisione trova un valido alleato nella farmacogenomica che mira a identificare varianti genetiche coinvolte nella risposta terapeutica. Nei trattamenti su misura, \ue8 necessaria un'attenzione particolare per quei farmaci che mostrano un basso indice terapeutico, come le fluoropirimidine (FL) che rappresentano la spina dorsale nella gestione di molti tipi di tumori solidi. FL portano a una grave grave tossicit\ue0 (G 653) in una percentuale considerevole di pazienti, causando occasionalmente la morte dei pazienti. Le FL sono metabolizzate principalmente dalla diidropirimidina deidrogenasi (DPD; DPYD) in metaboliti inattivi. Attualmente sono note solo quattro varianti genetiche della DPYD (DPYD * 2A, DPYD * 13, DPYD c.2846A> T e DPYD-HapB3) che riducono l'attivit\ue0 della DPD e di conseguenza sono associate alla tossicit\ue0 correlata alla FL. I quattro SNP utilizzati nel test di genotipizzazione sono raccomandati prima dell\u2019inizio di un trattamento basato su FL. Nonostante l'elevata specificit\ue0, questo test ha dimostrato una bassa sensibilit\ue0, lasciando inspiegabile la maggior parte degli eventi tossici. Recentemente, \ue8 emerso il ruolo precedentemente trascurato di nuove e rare varianti genetiche nella variabilit\ue0 intra-individuale nella risposta ai farmaci. In questa tesi di dottorato, abbiamo mirato a migliorare le conoscenze sulla validit\ue0 clinica e l'utilit\ue0 clinica del test diagnostico clinico DPYD proposto dalle attuali linee guida farmacogenomiche, studiando l'impatto delle quattro varianti della DPYD sulla tossicit\ue0 correlata alle fluoropirimidine e ai costi per la gestione della tossicit\ue0 in grandi coorti di pazienti. Inoltre, abbiamo analizzato il contributo di varianti genetiche nuove e rare in una serie di farmaco-geni nello sviluppo della tossicit\ue0 correlata alle fluoropirimidine. Qui, mostriamo come il test dei quattro SNP per DPYD consente di identificare un gran numero di pazienti che svilupperanno gravi reazioni avverse al trattamento a base di FL in un gruppo di 763 pazienti oncologici. Ci\uf2 \ue8 particolarmente vero se i quattro SNP DPYD sono disposti nel noto punteggio di attivit\ue0 genica (GAS) che pesa ciascun SNP in base al suo impatto specifico sul fenotipo della proteina DPD. Abbiamo anche dimostrato, in un sottogruppo di 550 pazienti con carcinoma del colon-retto, come il genotipo DPYD per il set di quattro SNP o il GAS siano correlati al costo di gestione per gravi tossicit\ue0 correlate alla FL. Questa scoperta ha notevoli implicazioni nell'utilit\ue0 del test che ne garantisce l'applicazione nella pratica clinica di routine. Successivamente abbiamo analizzato, con un approccio di sequenziamento di nuova generazione, il ruolo potenziale di varianti nuove o rare nei geni nella via di disintossicazione in 108 pazienti che non presentano le ben note 4 varianti di rischio DPYD e sviluppato grave tossicit\ue0 correlata al FL. Abbiamo identificato 9 rare varianti di DPYD, 3 erano missense molto rare e 6 erano missense uniche. In confronto, una popolazione di 106 pazienti trattati con un regime basato su FL, senza alcuna reazione avversa, ha mostrato una riduzione di nuove varianti (valore p = 0,001, secondo il test esatto di Fisher). Inoltre, come previsto, abbiamo trovato un arricchimento statisticamente significativo di varianti esoniche rare, molto rare e nuove sulla DPYD nella coorte di pazienti con grave tossicit\ue0 rispetto a quelle senza. Nonostante la necessit\ue0 clinica di personalizzare il trattamento su ciascun paziente, ad oggi tradurre le informazioni genetiche di ciascun paziente in un'indicazione terapeutica rimane un compito impegnativo. Lo studio della farmacogenomica nel trattamento a base di FL ha gi\ue0 fornito marcatori utili e validi per la pratica clinica, lo studio di varianti genetiche rare e nuove potrebbe in futuro migliorare ulteriormente gli strumenti disponibili per una medicina di precisione nel trattamento del cancro.Precision medicine finds a valuable ally in pharmacogenomics which is aimed at identifying genetic variants involved in therapeutic response. In tailored treatments, a specific attention is required for those drugs showing a low therapeutic index, such as fluoropyrimidines (FL) which represent the backbone in the management of many types of solid tumours. FL lead to severe toxicity (G 653) in a considerable percentage of patients, occasionally resulting in patients death. FL are mainly metabolized by dihydropyrimidine dehydrogenase (DPD; DPYD) into inactive metabolite. At present only four DPYD genetic variants (DPYD *2A, DPYD *13, DPYD c.2846A>T and DPYD-HapB3) are known to decrease DPD activity and consequently to be associated to FL-related toxicity. The four SNP genotyping test is recommended before an FL-based treatment by international shared pharmacogenetic guidelines, Despite the high specificity, this test demonstrated a low sensitivity, leaving the majority of the toxic events unexplained. Recently, the previously neglected role of novel and rare genetic variants in the intra-individual variability in the response to drugs has emerged. In this PhD thesis, we aimed at improving the knowledge on the clinical validity and clinical utility of the DPYD clinical diagnostic test proposed by the current pharmacogenomic guidelines, by investigating the impact of the four DPYD variants on fluoropyrimidines-related toxicity and costs for toxicity management in large cohorts of patients. In addition, we analysed the contribution of novel and rare genetic variants in a set of pharmaco-genes in the development of fluoropyrimidines-related toxicity. Here, we show how the four-SNPs test for DPYD allow to identify a large number of patients who will develop severe adverse reactions to FL-based treatment in a group of 763 oncological patients. This is especially true if the four DPYD SNPs are arranged in the so-called gene activity score (GAS) weighing each SNP according to its specific impact on the DPD protein phenotype. We also demonstrated, in a subset of 550 colorectal cancer patients, how the DPYD genotype for the four-SNPs set or the GAS are related to the management cost for severe toxicities related to FL. This finding has notable implication in the utility of the test warranting its application in the routine clinical practice. We subsequently analyzed, by a next generation sequencing approach, the potential role of novel or rare variants in genes in the detoxification pathway in 108 patients that are wild type for the well-know 4 DPYD risk variants and developed severe FL-related toxicity. We identified 9 rare DPYD variants, 3 were very rare missense (c.G345C, p.M115I; c.A2060C p.D687A and c.A2137G, p.N713D), and 6 were singleton missense (c.A110G, p.D37G; c.G481A, p.E161K; c.C800T, p.T267I; c.G958A, p.G320R; c.A1110G, p.I370M;c.C1579T, p.P527S). in comparison, a population of 106 patients treated with a FL-based regimen, without any adverse reaction, displayed a depletion of novel variants (p value =0.001, by Fisher\u2019s Exact Test). Also, as expected, we found a statistically significant enrichment of rare, very rare and novel exonic DPYD variants in the cohort of patients with severe toxicity as compared to those without (P =0.0187, P =0.0187, P =0.0291, respectively, , by Fisher\u2019s Exact Test). Despite the clinical need of tailoring treatment on each patient, to date translating the genetic information of each patient into a therapeutic indication remains a challenging task. The study of the pharmacogenomics in FL-based treatment has already provided useful and valid markers for the clinical practice, the study of rare and novel genetic variants could in the future further improve available tools for a precision medicine in cancer treatment

The use of pharmacogenetics to increase the safety of colorectal cancer patients treated with fluoropyrimidines

Fluoropyrimidines (FP) are given in the combination treatment of the advanced disease or as monotherapy in the neo-adjuvant and adjuvant treatment of colorectal cancerand other solid tumors including breast, head and neck and gastric cancer. FP present a narrow therapeutic index with 10 to 26% of patients experiencing acute severe or life-threatening toxicity. With the high number of patients receiving FP-based therapies, and the significant effects of toxicities on their quality of life, the prevention of FP-related adverse events is of major clinical interest. Host genetic variants in the rate limiting enzyme dihydropyrimidine dehydrogenase (DPYD) gene are related to the occurrence of extremely severe, early onset toxicity in FP treated patients. The pre-treatment diagnostic test of 4 DPYD genetic polymorphisms is suggested by the currently available pharmacogenetic guidelines. Several prospective implementation projects are ongoing to support the introduction of up-front genotyping of the patients in clinical practice. Multiple pharmacogenetic studies tried to assess the predictive role of other polymorphisms in genes involved in the FP pharmacokinetics/pharmacodynamic pathways, TYMS and MTHFR, but no additional clinically validated genetic markers of toxicity are available to date. The development of next-generation sequencing platforms opens new possibilities to highlight previously unreported genetic markers. Moreover, the investigation of the genetic variation in the patients immunological system, a pivotal target in cancer treatment, could bring notable advances in the field. This review will describe the most recent literature on the use of pharmacogenetics to increase the safety of a treatment based on FP administration in colorectal cancer patients

Virtual screening identifies a PIN1 inhibitor with possible antiovarian cancer effects

Peptidyl-prolyl cis–trans isomerase, NIMA-interacting 1 (PIN1) is a peptidyl-prolyl isomerase that binds phospho-Ser/Thr-Pro motifs in proteins and catalyzes the cis–trans isomerization of proline peptide bonds. PIN1 is overexpressed in several cancers including high-grade serous ovarian cancer. Since few therapies are effective against this cancer, PIN1 could be a therapeutic target but effective PIN1 inhibitors are lacking. To identify molecules with in vivo inhibitory effects on PIN1, we used consensus docking to model existing PIN1-ligand X-ray structures and to screen a chemical database for candidate inhibitors. Ten molecules were selected and tested in cellular assays, leading to the identification of VS10 that bound and inhibited PIN1. VS10 treatment reduced the viability of ovarian cancer cell lines by inducing proteasomal PIN1 degradation, without effects on PIN1 transcription, and also reduced the levels of downstream targets β-catenin, cyclin D1, and pSer473-Akt. VS10 is a selective PIN1 inhibitor that may offer new opportunities for treating PIN1-overexpressing tumors

Virtual screening identifies a PIN1 inhibitor with possible antiovarian cancer effects

Peptidyl-prolyl cis–trans isomerase, NIMA-interacting 1 (PIN1) is a peptidyl-prolyl isomerase that binds phospho-Ser/Thr-Pro motifs in proteins and catalyzes the cis–trans isomerization of proline peptide bonds. PIN1 is overexpressed in several cancers including high-grade serous ovarian cancer. Since few therapies are effective against this cancer, PIN1 could be a therapeutic target but effective PIN1 inhibitors are lacking. To identify molecules with in vivo inhibitory effects on PIN1, we used consensus docking to model existing PIN1-ligand X-ray structures and to screen a chemical database for candidate inhibitors. Ten molecules were selected and tested in cellular assays, leading to the identification of VS10 that bound and inhibited PIN1. VS10 treatment reduced the viability of ovarian cancer cell lines by inducing proteasomal PIN1 degradation, without effects on PIN1 transcription, and also reduced the levels of downstream targets β-catenin, cyclin D1, and pSer473-Akt. VS10 is a selective PIN1 inhibitor that may offer new opportunities for treating PIN1-overexpressing tumors

Rare genetic variant burden in DPYD predicts severe fluoropyrimidine-related toxicity risk

Preemptive targeted pharmacogenetic testing of candidate variations in DPYD is currently being used to limit toxicity associated with fluoropyrimidines. The use of innovative next generation sequencing (NGS) approaches could unveil additional rare (minor allele frequency <1%) genetic risk variants. However, their predictive value and management in clinical practice are still controversial, at least partly due to the challenges associated with functional analyses of rare variants. The aim of this study was to define the predictive power of rare DPYD variants burden on the risk of severe fluoropyrimidine-related toxicity. The DPYD coding sequence and untranslated regions were analyzed by NGS in 120 patients developing grade 3-5 (NCI-CTC vs3.0) fluoropyrimidine-related toxicity and 104 matched controls (no-toxicity). The functional impact of rare variants was assessed using two different in silico predictive tools (i.e., Predict2SNP and ADME Prediction Framework) and structural modeling. Plasma concentrations of uracil (U) and dihydrouracil (UH2) were quantified in carriers of the novel variants. Here, we demonstrate that the burden of rare variants was significantly higher in patients with toxicity compared to controls (p = 0.007, Mann-Whitney test). Carriers of at least one rare missense DPYD variant had a 16-fold increased risk in the first cycle and an 11-fold increased risk during the entire course of chemotherapy of developing a severe adverse event compared to controls (p = 0.013 and p = 0.0250, respectively by multinomial regression model). Quantification of plasmatic U/UH2 metabolites and in silico visualization of the encoded protein were consistent with the predicted functional effect for the novel variations. Analysis and consideration of rare variants by DPYD-sequencing could improve prevention of severe toxicity of fluoropyrimidines and improve patients' quality of life