Analisi farmacogenetica della timidilato sintetasi in pazienti affetti da carcinoma del colon-retto in trattamento con capecitabina: sviluppo di un nuovo metodo di genotipizzazione e applicazione clinica

Le fluoropirimidine sono inibitori della timidilato sintetasi (TS), enzima codificato da un gene che presenta, nella regione enhancer, ripetizioni contenenti l’upstream stimulatory factor-1 (USF-1) al quale si lega il fattore di trascrizione; tuttavia, la presenza di un polimorfismo G>C in USF-1 annulla la sua funzione. Poiché la quantità di TS cellulare influenza l’effetto delle fluoropirimidine, in questa tesi è stato validato un metodo di genotipizzazione in pazienti con carcinoma del colon-retto ed è stato correlato il numero di USF-1 con la tossicità delle fluoropirimidine. I risultati hanno dimostrato che la frequenza degli alleli 2RGC, 2RCC, 3RGGC e 3RGCC era 27,2, 20, 29,2 e 23,6%, rispettivamente. Le tossicità più frequenti erano nausea/vomito, diarrea, sindrome mano-piede e neutropenia e si manifestavano prevalentemente nei pazienti con numero di USF-1 ≤2. In conclusione, questo studio ha validato un metodo di genotipizzazione di TS e stabilito una correlazione tra tossicità e USF-1 boxes funzionali. Fluoropyrimidines are potent inhibitors of thymidylate synthase (TS), an enzyme encoded by a gene that displays, in the enhancer region, repetitions containing the upstream stimulatory factor-1 (USF-1) to which the transcription factor is bound. Since the cellular amount of TS modulates the effect of fluoropyrimidines, a genotyping method has been developed in this study and applied to patients affected by colorectal cancer and treated with fluoropyrimidines to investigate the possible relationship between the number of functional USF-1 boxes and the toxicity of treatment. Results showed that the allele frequencies of 2RGC, 2RCC, 3RGGC e 3RGCC were 27.2, 20, 29.2 and 23.6%. Most frequent toxicities were nausea/vomiting, diarrhea, hand-foot syndrome and neutropenia and occurred more frequently in patients with a number USF-1 boxes ≤2. In conclusion, this study validated a genotyping method of TS and established a correlation between toxicity and functional USF-1 boxes

Mathematical modeling of drug resistance due to KRAS mutation in colorectal cancer

The most challenging task in colorectal cancer research nowadays is to understand the development of acquired resistance to anti-EGFR drugs. The key reason for this problem is the KRAS mutations appearance after the treatment with monoclonal antibodies (moAb). Here we present a mathematical model for the analysis of KRAS mutations behavior in colorectal cancer with respect to moAb treatments. To evaluate the drug performance we have developed equations for two types of tumors cells, KRAS mutated and KRAS wild-type. Both tumor cell populations were treated with a combination of moAb and chemotherapy drugs. It was observed that even the minimal initial concentration of KRAS mutation before the treatment has the ability to make the tumor refractory to the treatment. Minor population of KRAS mutations has strong influence on large number of wild-type cells as well rendering them resistant to chemotherapy. Patient׳s immune responses are specifically taken into considerations and it is found that, in case of KRAS mutations, the immune strength does not affect medication efficacy. Finally, cetuximab (moAb) and irinotecan (chemotherapy) drugs are analyzed as first-line treatment of colorectal cancer with few KRAS mutated cells. Results show that this combined treatment could be only effective for patients with high immune strengths and it should not be recommended as first-line therapy for patients with moderate immune strengths or weak immune systems because of a potential risk of relapse, with KRAS mutant cells acquired resistance involved with them

Discovery of novel mutations in the dihydropyrimidine dehydrogenase gene associated with toxicity of fluoropyrimidines and viewpoint on preemptive pharmacogenetic screening in patients

Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the metabolic pathway of 5-fluorouracil (5-FU) and other fluoropyrimidines to inactive compounds. For this reason, severe, life-threatening toxicities may occur in patients with deficient DPD activity when administered standard doses of 5-FU and its prodrugs. Materials and methods: We selected three patients with colorectal adenocarcinoma who displayed unexpected severe adverse reactions after treatment with 5-FU and capecitabine. To investigate the possible involvement of deficient variants of the DPD gene (DPYD), a denaturing HPLC (dHPLC) approach followed by target exon sequencing of DPYD was performed on DNA extracted from peripheral blood. Results: Three novel non-synonymous mutations of DPYD, c.2509-2510insC, c.1801G>C, and c.680G>A, were detected in these subjects. Due to the absence of other deficient variants of DPYD and the compatibility of adverse reactions with fluoropyrimidine treatment, the novel variants were associated with a poor-metabolizer phenotype. Conclusions: Stratification of patients on the basis of their genotype may help prevent toxicity, and the large body of evidence about the pathogenesis of fluoropyrimidine-induced adverse reactions strongly encourages the adoption of best practice recommendations to appropriately address this important clinical issue. This approach is of utmost importance within a preventive, prognostic, and personalized approach to patient care in the oncology setting

Implications of KRAS mutations in acquired resistance to treatment in NSCLC

Rationale: KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies. The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients. Materials and Methods: A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance

Early changes in plasma DNA levels of mutant KRAS as a sensitive marker of response to chemotherapy in pancreatic cancer

Pancreatic cancer (PDAC) is still lacking of reliable markers to monitor tumor response. CA 19-9 is the only biomarker approved, despite it has several limitations in sensitivity and specificity. Since mutations of KRAS occur in more than 90% of tumors, its detection in circulating free tumor DNA (cftDNA) could represent a biomarker to monitor chemotherapy response. Twenty-seven advanced PDAC patients given first-line 5-fluorouracil, irinotecan and oxaliplatin or gemcitabine and nab-paclitaxel were enrolled. Three ml of plasma were collected: 1) before starting chemotherapy (baseline); 2) at day 15 of treatment; and 3) at each clinical follow-up. cftDNA was extracted and analysed for KRAS mutations (mutKRAS) by digital droplet PCR. Nineteen patients displayed a mutKRAS in baseline plasma samples. There was a statistically significant difference in progression-free survival (PFS) and overall survival (OS) in patients with increase vs. stability/reduction of cftDNA in the sample collected at day 15 (median PFS 2.5 vs 7.5 months, p = 0.03; median OS 6.5 vs 11.5 months, p = 0.009). The results of this study demonstrate that cftDNA mutKRAS changes are associated with tumor response to chemotherapy and support the evidence that mutKRAS in plasma may be used as a new marker for monitoring treatment outcome and disease progression in PDAC

Quantitative and Qualitative Analysis of Blood-based Liquid Biopsies to Inform Clinical Decision-making in Prostate Cancer

ADN tumoral circulant; Medicina de precisió; Càncer de pròstataADN tumoral circulante; Medicina de precisión; Cáncer de próstataCirculating tumor DNA; Precision medicine; Prostate cancerContext Genomic stratification can impact prostate cancer (PC) care through diagnostic, prognostic, and predictive biomarkers that aid in clinical decision-making. The temporal and spatial genomic heterogeneity of PC together with the challenges of acquiring metastatic tissue biopsies hinder implementation of tissue-based molecular profiling in routine clinical practice. Blood-based liquid biopsies are an attractive, minimally invasive alternative. Objective To review the clinical value of blood-based liquid biopsy assays in PC and identify potential applications to accelerate the development of precision medicine. Evidence acquisition A systematic review of PubMed/MEDLINE was performed to identify relevant literature on blood-based circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) in PC. Evidence synthesis Liquid biopsy has emerged as a practical tool to profile tumor dynamics over time, elucidating features that evolve (genome, epigenome, transcriptome, and proteome) with tumor progression. Liquid biopsy tests encompass analysis of DNA, RNA, and proteins that can be detected in CTCs, ctDNA, or EVs. Blood-based liquid biopsies have demonstrated promise in the context of localized tumors (diagnostic signatures, risk stratification, and disease monitoring) and advanced disease (response/resistance biomarkers and prognostic markers). Conclusions Liquid biopsies have value as a source of prognostic, predictive, and response biomarkers in PC. Most clinical applications have been developed in the advanced metastatic setting, where CTC and ctDNA yields are significantly higher. However, standardization of assays and analytical/clinical validation is necessary prior to clinical implementation

Application of a pharmacokinetic/pharmacogenetic approach to assess the nicotine metabolic profile of smokers in the real-life setting

The nicotine metabolite ratio, i.e., the ratio 3-hydroxycotinine/cotinine, is used to assess the nicotine metabolic status and has been proven to predict the response to smoking cessation treatments in randomized clinical trials. In the current study, a pharmacokinetic-pharmacogenetic integrated approach is described, based on the development of a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for nicotine metabolite ratio assay in plasma and a real-time PCR analysis for fast genotyping of CYP2A6. The pharmacokinetic-pharmacogenetic approach was validated in 66 subjects with different smoking status. The LC/MS/MS assay was rapid and sensitive enough to detect plasma cotinine levels also in second-hand exposed abstainers. In the cohort of patients of the present study the following results were obtained: (i) the frequencies of CYP2A6 genetic variants were comparable with those from clinical trials carried out in Caucasian populations; (ii) all the subjects carrying the CYP2A6 deficient allele also had a slow metabolizer phenotype; (iii) slow metabolizers had mean nicotine metabolite ratio approximately 50% of that of the normal/fast metabolizers; (iv) women had higher nicotine metabolite ratio than men; and (v) salivary nicotine metabolite ratio measures were comparable to plasma levels. Overall, the findings of the current study demonstrate that the simultaneous assessment of nicotine metabolite ratio and CYP2A6 genotype from human blood samples is feasible and accurate and could be used in a smoking cessation program to optimize treatments and identify those smokers who inherit metabolically deficient CYP2A6 alleles

Concise Review: Chronic Myeloid Leukemia: Stem Cell Niche and Response to Pharmacologic Treatment

Nowadays, more than 90% of patients affected by chronic myeloid leukemia (CML) survive with a good quality of life, thanks to the clinical efficacy of tyrosine kinase inhibitors (TKIs). Nevertheless, point mutations of the ABL1 pocket occurring during treatment may reduce binding of TKIs, being responsible of about 20% of cases of resistance among CML patients. In addition, the presence of leukemic stem cells (LSCs) represents the most important event in leukemia progression related to TKI resistance. LSCs express stem cell markers, including active efflux pumps and genetic and epigenetic alterations together with deregulated cell signaling pathways involved in self-renewal, such as Wnt/β-catenin, Notch, and Hedgehog. Moreover, the interaction with the bone marrow microenvironment, also known as hematopoietic niche, may influence the phenotype of surrounding cells, which evade mechanisms controlling cell proliferation and are less sensitive or frankly resistant to TKIs. This Review focuses on the role of LSCs and stem cell niche in relation to response to pharmacological treatments. A literature search from PubMed database was performed until April 30, 2017, and it has been analyzed according to keywords such as chronic myeloid leukemia, stem cell, leukemic stem cells, hematopoietic niche, tyrosine kinase inhibitors, and drug resistance. Stem Cells Translational Medicine 2018

Benefits and Harms of Lung Cancer Screening by Chest Computed Tomography: A Systematic Review and Meta-Analysis

PURPOSE This meta-analysis aims to combine and analyze randomized clinical trials comparing computed tomography lung screening (CTLS) versus either no screening (NS) or chest x-ray (CXR) in subjects with cigarette smoking history, to provide a precise and reliable estimation of the benefits and harms associated with CTLS. MATERIALS AND METHODS Data from all published randomized trials comparing CTLS versus either NS or CXR in a highly tobacco-exposed population were collected, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Subgroup analyses by comparator (NS or CXR) were performed. Pooled risk ratio (RR) and relative 95% CIs were calculated for dichotomous outcomes. The certainty of the evidence was assessed using the GRADE approach. RESULTS Nine eligible trials (88,497 patients) were included. Pooled analysis showed that CTLS is associated with: a significant reduction of lung cancer-related mortality (overall RR, 0.87; 95% CI, 0.78 to 0.98; NS RR, 0.80; 95% CI, 0.69 to 0.92); a significant increase of early-stage tumors diagnosis (overall RR, 2.84; 95% CI 1.76 to 4.58; NS RR, 3.33; 95% CI, 2.27 to 4.89; CXR RR, 1.52; 95% CI, 1.04 to 2.23); a significant decrease of late-stage tumors diagnosis (overall RR, 0.75; 95% CI, 0.68 to 0.83; NS RR, 0.67; 95% CI, 0.56 to 0.80); a significant increase of resectability rate (NS RR, 2.57; 95% CI, 1.76 to 3.74); a nonsignificant reduction of all-cause mortality (overall RR, 0.99; 95% CI, 0.94 to 1.05); and a significant increase of overdiagnosis rate (NS, 38%; 95% CI, 14 to 63). The analysis of lung cancer-related mortality by sex revealed nonsignificant differences between men and women (P = .21; I-squared = 33.6%). CONCLUSION Despite there still being uncertainty about overdiagnosis estimate, this meta-analysis suggested that the CTLS benefits outweigh harms, in subjects with cigarette smoking history, ultimately supporting the systematic implementation of lung cancer screening worldwide