Innovative strategies for tailoring therapy in cancer patient: pharmacogenetics and hormone therapy personalization in metastatic or locally advanced breast cancer patients treated with Exemestane

Background: Response to chemotherapeutic agents is highly variable among patients both in terms of efficacy and tolerability; consequently personalization of drug therapy is one of the main objective in cancer treatment in order to reduce adverse drug reactions (ADRs), improve efficacy while decreasing the costs of treatments. Many factors account for inter-individual differences. Among them, patient’s genetic background has attracted interest for personalization of drug therapy (Pharmacogenetics). Breast cancer (BC) is the female most frequently diagnosed malignancy and the primary cause of cancer-related death among females with 1.380.000 new cases and 458.000 deaths worldwide registered in 2008. Estrogen receptors (ER) are over-expressed in around 80% of breast cancer cases and ER-positive (ER+) cancer cells depend on estrogens for their growth. In postmenopausal women estrogens can derive only from androgens through an aromatization reaction. Aromatase (CYP19A1) is a key enzyme in this process and, for this reason, is the target of many inhibitors drugs, including exemestane. Anti-aromatase treatments represent the current corner-stone of ER+ BC therapy in postmenopausal women. Exemestane is a steroidal irreversible third generation aromatase inhibitor (AI) which determines the inactivation of the enzyme, resulting in estrogen synthesis inhibition and deprivation. Exemestane is used in adjuvant setting for ER+ early stage invasive BC and for the treatment of advanced stage BC progressed to a previous anti-estrogen therapy. Several germ line variations (polymorphisms) have been described in genes involved either in estrogens activity and metabolism or in the pharmacokinetics of exemestane. Aims: this PhD thesis had a dual aim: * setting up a pharmacogenetic method to analyze estrogen and exemestane-related polymorphisms (Single Nucleotide Polymorphisms (SNPs) and Short Tandem Repeats (STRs)), * determining the predictive and prognostic value of these polymorphisms in postmenopausal metastatic or locally advanced ER+ BC patients (Response Rate (RR), Clinical Benefit (CB), Time To Progression (TTP) and Overall Survival (OS)). Fifteen polymorphisms in genes involved estrogens synthesis (CYP17A1 and CYP19A1), activity (ESR1, ESR2 and RIZ1) and metabolism (CYP1B1, UGT1A1 and COMT) as well as genes implicated in the metabolic pathway of exemestane (CYP3A4 and CYP3A5) were investigated. We considered the CYP19A1_Ex11_410A/C (rs4646) SNP in the 3’ untraslated region (3'UTR) of the aromatase gene, previously associated to a better disease-free survival (DFS) (Colomer et al., 2008) in patients treated with the AI, letrozole, and to a better OS in patients treated with another AI, anastrozole (Liu et al., 2013). However, the role of this polymorphism has not yet been clearly defined. Other CYP19A1 polymorphisms analyzed were: CYP19A1_47T/C (rs700519), CYP19A1_1558T/C (rs10046) and CYP19A1_(TTTA)n (rs60271534), along with a SNP on the CYP17A1 gene (CYP17A1_27A/G (rs743572)), coding for another enzyme responsible for estrogens synthesis. Additionally, we investigated: * polymorphisms on estrogens receptors: ESR1: ESR1_497T/C (rs2234693), and ESR1_256A/G (rs9340799); ESR2: ESR2_1082A/G (rs1256049), and ESR2_1730A/G (rs4986938); RIZ1: RIZ1_delP704 (rs2308040); * polymorphisms on estrogens metabolizing enzymes: CYP1B1: CYP1B1*3_4326G/C (rs1056836), UGT1A1: UGT1A1*28_ TA(6/7) (rs8175347) and COMT: COMT_12A/G (rs4680); * polymorphisms on enzymes involved in the oxidative metabolism of exemestane: CYP3A4: CYP3A4*1B_-392A/G (rs2740574) and CYP3A5: CYP3A5*3_6986A/G (rs776746). Methods: genetic analyses were conducted in a group of 275 ER+ metastatic or locally advanced BC patients treated with exemestane as first line hormone therapy. Patients were subjected to blood sampling before the beginning of therapy. DNA was extracted from whole blood, and then amplified by Polymerase Chain Reaction (PCR). Four methods for polymorphisms genotyping were set up and developed: Pyrosequencing, TaqMan® Allelic Discrimination Assay, Automated Fragment Analysis and Illumina GoldenGate Assay. Statistical associations between genetic determinants and clinical outcome were assessed by the two-sided Fisher’s Exact Test (associations between genotypes and clinical responses) and the Kaplan-Meier product-limit method with the log-rank test statistic (associations between polymorphisms and TTP/OS). Results: For each polymorphism the most appropriate technique, based on the best result obtained in the setting up process, was chosen. As a result: * three SNPs were investigated with Pyrosequencing: CYP19A1_47T/C (rs700519), CYP3A4*1B_-392A/G (rs2740574), and RIZ1_delP704 (rs2308040); * ten SNPs were genotyped with TaqMan® Allelic Discrimination Assay: CYP19A1_Ex11+410A/C (rs4646), CYP19A1_1558T/C (rs10046), CYP3A5*3_6986A/G (rs776746), COMT_12A/G (rs4680), ESR1_497T/C (rs2234693), ESR1_256A/G (rs9340799), ESR2_1082A/G (rs1256049), ESR2_1730A/G (rs4986938), CYP17A1_27A/G (rs743572) and CYP1B1*3_4326G/C (rs1056836); * two STR were analyzed with Automated Fragment Analysis: CYP19A1_(TTTA)n (rs60271534) and UGT1A1*28_ TA(6/7) (rs8175347) * twelve of the above mentioned polymorphisms were additionally analyzed by Illumina GoldenGate Assay as positive controls. The results obtained by this validation process was a 100% accordance within the genotypes obtained. Among the polymorphisms investigated, a statistically significant association was observed for CYP1B1, the gene encoding for the enzyme which catalyze the phase I estrogens oxidative metabolism. The variant (G) allele of CYP1B1*3_4326G/C (rs1056836) was significantly associated with clinical response to exemestane (RR, ORGG = 2.91, 95% CI = 5.88 – 1.25, p = 0.0039; according to the two-sided Fisher’s exact test). The same variant allele was also significantly associated with the TTP and OS (TTP, dominant model: HR CG+GG= 0.66, 95% CI = 0.50 – 0.87, p = 0.0037; OS, dominant model = HR CG+GG= 0.66, 95% CI = 0.46 – 0.95, p = 0.023, according to the log-rank test) meaning that patients carrying at least one variant allele (G) not only showed a better clinical response, but experienced also a later progression and a longer survival than wild type patients. Regarding the aromatase gene (CYP19A1 gene), the only association found, even if marginal, was between CYP19A1_1558T/C (rs10046) SNP and TTP (HRCC recessive model=1.4, 95%CI = 1.04 – 1.89, p = 0.028). Conversely, we did not find any significant association between CYP19A1_Ex11_410A/C (rs4646) SNP, (the main objective of the study) and RR, CB, TTP or OS, respectively. Concerning aromatase gene polymorphisms, we were able to describe a new genetic variant for the CYP19A1_(TTTA)n (rs60271534) STR in intron 4. Genetic databases and literature report that the number of repeats varies from 7 to 13, but we found a still not described 14 (TTTA) repeats allele. Conclusions: in conclusion, this thesis work allowed defining a new molecular marker, CYP1B1*3_4326G/C (rs1056836) SNP, with a predictive and prognostic value for the exemestane-based treatment of postmenopausal ER+ metastatic or locally advanced BC patients. This indicates that, once validated, this marker could potentially be employable in the daily clinical oncology practice as a tool which may allow the identification of patients more likely to be responsive to treatment by a simple genetic evaluation from peripheral blood, performed prior to therapy. In addition, we described a new genetic variant in the aromatase gene

Population pharmacokinetic modelling of imatinib in healthy subjects receiving a single dose of 400 mg

Purpose: Imatinib is indicated for treatment of CML, GIST, etc. The population pharmacokinetics (popPK) of imatinib in patients under long-term treatment are reported in literature. Data obtained from bioequivalence trials for healthy subjects were used to evaluate the influence of demographic and pharmacogenetic factors on imatinib pharmacokinetics (PK) in a collective without concurrent drugs, organ dysfunction, inflammation etc. In addition, the differences in PK between the healthy subjects and a patient cohort was examined to identify possible disease effects. Methods: 26 volunteers were administered orally with single dose of 400 mg imatinib. 16–19 plasma samples per volunteer were collected from 0.5 up to 72 h post-dose. The popPK was built and post hoc estimates were compared with previously published PK parameters evaluated by non-compartmental analysis in the same cohort. The predictivity of the model for data collected from 40 patients with gastrointestinal stromal tumors at steady state was evaluated. Results: The popPK was best described by a two-compartment transit model with first-order elimination. No significant covariates were identified, probably due to the small cohort and the narrow range of demographic covariates; CYP3A5 phenotypes appeared to have some influence on the clearance of imatinib. Good agreement between non-compartment and popPK analyses was observed with the differences of the geometric means/ median of PK estimates below 10%. The model indicated lower clearance for patients compared to healthy volunteers (p value < 0.01). Conclusion: The two-compartment transit model adequately describes the absorption and distribution of imatinib in healthy volunteers. For patients, a lower clearance of imatinib compared to healthy volunteer was estimated by the model. The model can be applied for dose individualization based on trough concentrations assuming no significant differences in absorption between patients and healthy volunteersThis work was part of the master these of Yi-Han Chien. There was no funding for this work. P. Zubiaur’s contract with CIBERehd is fnanced by the “Infraestructura de Medicina de Precisión asociada a la Ciencia y Tecnología (IMPaCT, IMP/00009)”, Instituto de Salud Carlos III (ISCIII

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

IL15RA and SMAD3 Genetic Variants Predict Overall Survival in Metastatic Colorectal Cancer Patients Treated with FOLFIRI Therapy: A New Paradigm

Simple SummaryThere is an increasing scientific interest in the study of the interaction between the immune system and drugs in cancer that can affect the efficacy of an anti-cancer treatment. This study was undertaken to better understand if the genetic characteristic of a cancer patient's immune system can predict the tumor response to the treatment and the duration of survival. The topic was studied on 335 metastatic colorectal cancer patients treated with a first-line chemotherapy (FOLFIRI regimen, irinotecan-5-fluorouracil-leucovorin). The research highlighted two markers, IL15RA-rs7910212 and SMAD3-rs7179840, significantly associated with the patient's survival. When considering IL15RA-rs7910212 and SMAD3-rs7179840 in combination with other two genetic markers previously investigated (NR1I2-rs1054190, VDR-rs7299460), we built up a highly predictive genetic score of survival. The herein identified markers must be further validated, but still represent good candidates to understand how much a patient with a metastatic colorectal cancer can benefit from a chemotherapy with FOLFIRI regimen.A new paradigm in cancer chemotherapy derives from the interaction between chemotherapeutics, including irinotecan and 5-fluorouracil (5-FU), and the immune system. The patient's immune response can modulate chemotherapy effectiveness, and, on the other hand, chemotherapeutic agents can foster tumor cell immunogenicity. On these grounds, the analysis of the cancer patients' immunogenetic characteristics and their effect on survival after chemotherapy represent a new frontier. This study aims to identify genetic determinants in the immuno-related pathways predictive of overall survival (OS) after FOLFIRI (irinotecan, 5-FU, leucovorin) therapy. Two independent cohorts comprising a total of 335 patients with metastatic colorectal cancer (mCRC) homogeneously treated with first-line FOLFIRI were included in the study. The prognostic effect of 192 tagging genetic polymorphisms in 34 immune-related genes was evaluated using the bead array technology. The IL15RA rs7910212-C allele was associated with worse OS in both discovery (HR: 1.57, p = 0.0327, Bootstrap p-value = 0.0280) and replication (HR: 1.71, p = 0.0411) cohorts. Conversely, SMAD3 rs7179840-C allele was associated with better OS in both discovery (HR: 0.65, p = 0.0202, Bootstrap p-value = 0.0203) and replication (HR: 0.61, p = 0.0216) cohorts. A genetic prognostic score was generated integrating IL15RA-rs7910212 and SMAD3-rs7179840 markers with inflammation-related prognostic polymorphisms we previously identified in the same study population (i.e., PXR [NR1I2]-rs1054190, VDR-rs7299460). The calculated genetic score successfully discriminated patients with different survival probabilities (p < 0.0001 log-rank test). These findings provide new insight on the prognostic value of genetic determinants, such as IL15RA and SMAD3 markers, and could offer a new decision tool to improve the clinical management of patients with mCRC receiving FOLFIRI

A New Genetic Risk Score to Predict the Outcome of Locally Advanced or Metastatic Breast Cancer Patients Treated With First-Line Exemestane: Results From a Prospective Study

Currently there are no reliable biomarkers to predict outcome of exemestane treatment. We designed a prospective study to investigate whether constitutive genetic background might affect response to therapy. In a population of 302 advanced breast cancer patients treated with exemestane we showed that a 5-polymorphism-based genetic score could be used to identify patients with different risks of progression and death.Introduction: Approximately 50% of locally advanced or metastatic breast cancer (MBC) patients treated with first-line exemestane do not show objective response and currently there are no reliable biomarkers to predict the outcome of patients using this therapy. The constitutive genetic background might be responsible for differences in the outcome of exemestane-treated patients. We designed a prospective study to investigate the role of germ line polymorphisms as biomarkers of survival. Patients and Methods: Three hundred two locally advanced or MBC patients treated with first-line exemestane were genotyped for 74 germ line polymorphisms in 39 candidate genes involved in drug activity, hormone balance, DNA replication and repair, and cell signaling pathways. Associations with progression-free survival (PFS) and overall survival (OS) were tested with multivariate Cox regression. Bootstrap resampling was used as an internal assessment of results reproducibility. Results: Cytochrome P450 19A1-rs10046TC/CC, solute carrier organic anion transporter 1B1-rs4149056TT, adenosine triphosphate binding cassette subfamily G member 2-rs2046134GG, fibroblast growth factor receptor-4-rs351855TT, and X-ray repair cross complementing 3-rs861539TT were significantly associated with PFS and then combined into a risk score (0-1, 2, 3, or 4-6 risk points). Patients with the highest risk score (4-6 risk points) compared with ones with the lowest score (0-1 risk points) had a median PFS of 10 months versus 26.3 months (adjusted hazard ratio [AdjHR], 3.12 [95% confidence interval (CI), 2.18-4.48]; P < .001) and a median OS of 38.9 months versus 63.0 months (AdjHR, 2.41 [95% CI, 1.22-4.79], P = .012), respectively. Conclusion: In this study we defined a score including 5 polymorphisms to stratify patients for PFS and OS. This score, if validated, might be translated to personalize locally advanced or MBC patient treatment and management

Innovative strategies for tailoring therapy in cancer patient: pharmacogenetics and hormone therapy personalization in metastatic or locally advanced breast cancer patients treated with Exemestane

Background: Response to chemotherapeutic agents is highly variable among patients both in terms of efficacy and tolerability; consequently personalization of drug therapy is one of the main objective in cancer treatment in order to reduce adverse drug reactions (ADRs), improve efficacy while decreasing the costs of treatments. Many factors account for inter-individual differences. Among them, patient’s genetic background has attracted interest for personalization of drug therapy (Pharmacogenetics). Breast cancer (BC) is the female most frequently diagnosed malignancy and the primary cause of cancer-related death among females with 1.380.000 new cases and 458.000 deaths worldwide registered in 2008. Estrogen receptors (ER) are over-expressed in around 80% of breast cancer cases and ER-positive (ER+) cancer cells depend on estrogens for their growth. In postmenopausal women estrogens can derive only from androgens through an aromatization reaction. Aromatase (CYP19A1) is a key enzyme in this process and, for this reason, is the target of many inhibitors drugs, including exemestane. Anti-aromatase treatments represent the current corner-stone of ER+ BC therapy in postmenopausal women. Exemestane is a steroidal irreversible third generation aromatase inhibitor (AI) which determines the inactivation of the enzyme, resulting in estrogen synthesis inhibition and deprivation. Exemestane is used in adjuvant setting for ER+ early stage invasive BC and for the treatment of advanced stage BC progressed to a previous anti-estrogen therapy. Several germ line variations (polymorphisms) have been described in genes involved either in estrogens activity and metabolism or in the pharmacokinetics of exemestane. Aims: this PhD thesis had a dual aim: * setting up a pharmacogenetic method to analyze estrogen and exemestane-related polymorphisms (Single Nucleotide Polymorphisms (SNPs) and Short Tandem Repeats (STRs)), * determining the predictive and prognostic value of these polymorphisms in postmenopausal metastatic or locally advanced ER+ BC patients (Response Rate (RR), Clinical Benefit (CB), Time To Progression (TTP) and Overall Survival (OS)). Fifteen polymorphisms in genes involved estrogens synthesis (CYP17A1 and CYP19A1), activity (ESR1, ESR2 and RIZ1) and metabolism (CYP1B1, UGT1A1 and COMT) as well as genes implicated in the metabolic pathway of exemestane (CYP3A4 and CYP3A5) were investigated. We considered the CYP19A1_Ex11_410A/C (rs4646) SNP in the 3’ untraslated region (3'UTR) of the aromatase gene, previously associated to a better disease-free survival (DFS) (Colomer et al., 2008) in patients treated with the AI, letrozole, and to a better OS in patients treated with another AI, anastrozole (Liu et al., 2013). However, the role of this polymorphism has not yet been clearly defined. Other CYP19A1 polymorphisms analyzed were: CYP19A1_47T/C (rs700519), CYP19A1_1558T/C (rs10046) and CYP19A1_(TTTA)n (rs60271534), along with a SNP on the CYP17A1 gene (CYP17A1_27A/G (rs743572)), coding for another enzyme responsible for estrogens synthesis. Additionally, we investigated: * polymorphisms on estrogens receptors: ESR1: ESR1_497T/C (rs2234693), and ESR1_256A/G (rs9340799); ESR2: ESR2_1082A/G (rs1256049), and ESR2_1730A/G (rs4986938); RIZ1: RIZ1_delP704 (rs2308040); * polymorphisms on estrogens metabolizing enzymes: CYP1B1: CYP1B1*3_4326G/C (rs1056836), UGT1A1: UGT1A1*28_ TA(6/7) (rs8175347) and COMT: COMT_12A/G (rs4680); * polymorphisms on enzymes involved in the oxidative metabolism of exemestane: CYP3A4: CYP3A4*1B_-392A/G (rs2740574) and CYP3A5: CYP3A5*3_6986A/G (rs776746). Methods: genetic analyses were conducted in a group of 275 ER+ metastatic or locally advanced BC patients treated with exemestane as first line hormone therapy. Patients were subjected to blood sampling before the beginning of therapy. DNA was extracted from whole blood, and then amplified by Polymerase Chain Reaction (PCR). Four methods for polymorphisms genotyping were set up and developed: Pyrosequencing, TaqMan® Allelic Discrimination Assay, Automated Fragment Analysis and Illumina GoldenGate Assay. Statistical associations between genetic determinants and clinical outcome were assessed by the two-sided Fisher’s Exact Test (associations between genotypes and clinical responses) and the Kaplan-Meier product-limit method with the log-rank test statistic (associations between polymorphisms and TTP/OS). Results: For each polymorphism the most appropriate technique, based on the best result obtained in the setting up process, was chosen. As a result: * three SNPs were investigated with Pyrosequencing: CYP19A1_47T/C (rs700519), CYP3A4*1B_-392A/G (rs2740574), and RIZ1_delP704 (rs2308040); * ten SNPs were genotyped with TaqMan® Allelic Discrimination Assay: CYP19A1_Ex11+410A/C (rs4646), CYP19A1_1558T/C (rs10046), CYP3A5*3_6986A/G (rs776746), COMT_12A/G (rs4680), ESR1_497T/C (rs2234693), ESR1_256A/G (rs9340799), ESR2_1082A/G (rs1256049), ESR2_1730A/G (rs4986938), CYP17A1_27A/G (rs743572) and CYP1B1*3_4326G/C (rs1056836); * two STR were analyzed with Automated Fragment Analysis: CYP19A1_(TTTA)n (rs60271534) and UGT1A1*28_ TA(6/7) (rs8175347) * twelve of the above mentioned polymorphisms were additionally analyzed by Illumina GoldenGate Assay as positive controls. The results obtained by this validation process was a 100% accordance within the genotypes obtained. Among the polymorphisms investigated, a statistically significant association was observed for CYP1B1, the gene encoding for the enzyme which catalyze the phase I estrogens oxidative metabolism. The variant (G) allele of CYP1B1*3_4326G/C (rs1056836) was significantly associated with clinical response to exemestane (RR, ORGG = 2.91, 95% CI = 5.88 – 1.25, p = 0.0039; according to the two-sided Fisher’s exact test). The same variant allele was also significantly associated with the TTP and OS (TTP, dominant model: HR CG+GG= 0.66, 95% CI = 0.50 – 0.87, p = 0.0037; OS, dominant model = HR CG+GG= 0.66, 95% CI = 0.46 – 0.95, p = 0.023, according to the log-rank test) meaning that patients carrying at least one variant allele (G) not only showed a better clinical response, but experienced also a later progression and a longer survival than wild type patients. Regarding the aromatase gene (CYP19A1 gene), the only association found, even if marginal, was between CYP19A1_1558T/C (rs10046) SNP and TTP (HRCC recessive model=1.4, 95%CI = 1.04 – 1.89, p = 0.028). Conversely, we did not find any significant association between CYP19A1_Ex11_410A/C (rs4646) SNP, (the main objective of the study) and RR, CB, TTP or OS, respectively. Concerning aromatase gene polymorphisms, we were able to describe a new genetic variant for the CYP19A1_(TTTA)n (rs60271534) STR in intron 4. Genetic databases and literature report that the number of repeats varies from 7 to 13, but we found a still not described 14 (TTTA) repeats allele. Conclusions: in conclusion, this thesis work allowed defining a new molecular marker, CYP1B1*3_4326G/C (rs1056836) SNP, with a predictive and prognostic value for the exemestane-based treatment of postmenopausal ER+ metastatic or locally advanced BC patients. This indicates that, once validated, this marker could potentially be employable in the daily clinical oncology practice as a tool which may allow the identification of patients more likely to be responsive to treatment by a simple genetic evaluation from peripheral blood, performed prior to therapy. In addition, we described a new genetic variant in the aromatase gene.Introduzione: La risposta agli agenti chemioterapici è altamente variabile tra i pazienti sia per quanto riguarda l’efficacia che la tollerabilità, di conseguenza la personalizzazione della terapia è uno dei principali obiettivi della ricerca in campo oncologico con l’obiettivo di ridurre le reazioni avverse al farmaco, migliorarne l’efficacia e nel contempo contenerne i costi. I fattori responsabili della variabilità interindividuale sono molteplici. Tra questi, il background genetico dei pazienti ha attratto interesse per la personalizzazione della terapia (Farmacogenetica). Il carcinoma mammario (breast cancer - BC) rappresenta la neoplasia più frequentemente diagnosticata e la prima causa di morte collegata al cancro tra le donne. Nel 2008 sono stati registrati, a livello mondiale, 1.380.000 nuovi casi e 458.000 morti a causa del cancro della mammella. Il recettore degli estrogeni (estrogen receptor - ER) risulta iper-espresso in circa l’80% dei casi di BC e le cellule cancerose positive al ER (ER+) dipendono dagli estrogeni per la loro crescita. Nelle donne in menopausa, gli estrogeni derivano unicamente dagli androgeni attraverso una reazione di aromatizzazione. L’aromatasi (CYP19A1) è un enzima chiave in questo processo e, per questa ragione, è diventato il target di numerosi farmaci inibitori, compreso exemestane. Il trattamento anti-aromatasi rappresenta attualmente il cardine della terapia del ER+ BC nelle donne in menopausa. Exemestane è un inibitore irreversibile dell’aromatasi (AI) di terza generazione e di tipo steroideo che determina l’inattivazione dell’enzima, provocando quindi l’inibizione della sintesi estrogenica. Exemestane è un farmaco impiegato in assetto adiuvante per il ER+ BC invasivo allo stadio precoce ed in assetto avanzato se la malattia è progredita dopo una precedente terapia anti-estrogenica. Sono state descritte numerose variazioni genetiche germinali (polimorfismi) in geni coinvolti sia nell’attività e metabolismo degli estrogeni che nella farmacocinetica di exemestane. Obiettivi: questa tesi di dottorato ha avuto un duplice obiettivo: * mettere a punto un metodo di indagine farmacogenetica per analizzare polimorfismi correlati ad estrogeni ed exemestane (polimorfismi a singolo nucleotide – SNPs e microsatelliti (short tandem repeats) - STRs) * determinare il ruolo predittivo e prognostico di tali polimorfismi come biomarcatori di efficacia del trattamento a base di exemestane, in termini di Response Rate (RR), Clinical Benefit (CB), tempo alla progressione (TTP) e sopravvivenza globale (OS). Sono stati considerati quindici polimorfismi in geni coinvolti nella sintesi (CYP17A1 e CYP19A1), attività (ESR1, ESR2 e RIZ1) e metabolismo (CYP1B1, UGT1A1 e COMT) degli estrogeni insieme a geni implicati nel pathway metabolico di exemestane (CYP3A4 e CYP3A5). Come obiettivo primario dello studio clinico è stato considerato lo SNP CYP19A1_Ex11_410A/C (rs4646) della regione non 3’ tradotta (3’ untraslated region - 3’UTR) del gene dell’aromatasi, già in precedenza associato ad una migliore sopravvivenza libera da malattia (disease free servival – DFS) (Colomer et al., 2008) in pazienti trattate con l’AI letrozolo e con la miglior OS in pazienti trattate con un altro AI, l’anastrozolo (Liu et al., 2013). Ciononostante, il ruolo di questo polimorfismo non è stato ancora chiaramente definito. Sono stati analizzati anche altri polimorfismi del gene CYP19A1 (CYP19A1_47T/C (rs700519), CYP19A1_1558T/C (rs10046) e CYP19A1_(TTTA)n (rs60271534)) insieme ad uno SNP nel gene CYP17A1(CYP17A1_27A/G (rs743572)), codificante per un altro enzima responsabile della sintesi degli estrogeni. Inoltre, sono stati indagati polimorfismi dei geni codificanti per: * i recettori degli estrogeni: ESR1: ESR1_497T/C (rs2234693), e ESR1_256A/G (rs9340799); ESR2: ESR2_1082A/G (rs1256049), e ESR2_1730A/G (rs4986938); RIZ1: RIZ1_delP704 (rs2308040); * gli enzimi deputati al metabolismo degli estrogeni: CYP1B1: CYP1B1*3_4326G/C (rs1056836), UGT1A1: UGT1A1*28_ TA(6/7) (rs8175347) e COMT: COMT_12A/G (rs4680); * gli enzimi responsabili del metabolismo ossidativo di exemestane: CYP3A4: CYP3A4*1B_-392A/G (rs2740574) e CYP3A5: CYP3A5*3_6986A/G (rs776746). Metodi: le analisi genetiche sono state condotte in un gruppo di 275 pazienti affetti da ER+ BC metastatico o localmente avanzato trattate con exemestane come prima linea di trattamento ormonale. Ai pazienti è stato effettuato un prelievo ematico prima dell’inizio della terapia. Il DNA è stato poi estratto dal campione di sangue intero ed amplificato tramite la reazione a catena della polimerasi (PCR). Per le analisi genetiche sono state messe a punto quattro tecniche di genotipizzazione: Pyrosequencing, Saggio di Discriminazione Allelica mediante sonde TaqMan®, Analisi dei Frammenti Automatizzata ed il saggio GoldenGate di Illumina. Sono state valutate le associazioni statistiche tra i determinanti genetici e l’outcome clinico dei pazienti attraverso il Test Esatto di Fisher a due vie per l’associazione di polimorfismi e risposta clinica e attraverso lo stimatore del prodotto limite di Kaplan Meier e il test dei ranghi logaritmici per l’associazione tra polimorfismi e TTP/OS. Risultati: per ogni polimorfismo è stata scelta la tecnica di indagine molecolare più appropriata a seconda del miglior risultato ottenuto durante la fase di messa a punto delle metodologie. Di conseguenza: * tre SNPs sono stati analizzati con il Pyrosequencing: CYP19A1_47T/C (rs700519), CYP3A4*1B_-392A/G (rs2740574), e RIZ1_delP704 (rs2308040); * dieci SNPs sono stati genotipizzati con il saggio di Discriminazione Allelica mediante sonde TaqMan®: CYP19A1_Ex11+410A/C (rs4646), CYP19A1_1558T/C (rs10046), CYP3A5*3_6986A/G (rs776746), COMT_12A/G (rs4680), ESR1_497T/C (rs2234693), ESR1_256A/G (rs9340799), ESR2_1082A/G (rs1256049), ESR2_1730A/G (rs4986938), CYP17A1_27A/G (rs743572) e CYP1B1*3_4326G/C (rs1056836); * due STR sono stati esaminati attraverso l’Analisi dei Frammenti Automatizzata: CYP19A1_(TTTA)n (rs60271534) e UGT1A1*28_ TA(6/7) (rs8175347); * i campioni analizzati per dodici dei sopraccitati polimorfismi sono stati, inoltre, inclusi nel saggio Illumina GoldenGate come controlli positivi. Il risultato di questo processo di validazione è stata una concordanza del 100% tra i genotipi ottenuti con questa tecnica e quelli derivanti dalle precedenti indagini. Tra i polimorfismi analizzati, è stata osservata un’associazione statisticamente significativa per CYP1B1, gene codificante per l’enzima responsabile del metabolismo ossidativo di prima fase degli estrogeni. L’allele variante G del polimorfismo CYP1B1*3_4326G/C (rs1056836) è stato significativamente associato con la risposta clinica ad exemestane (RR, ORGG = 2.91, 95% CI = 5.88 – 1.25, p = 0.0039; secondo il Test Esatto di Fisher a due vie). Lo stesso allele variante è stato significativamente associato anche al TTP e alla OS (TTP, modello dominante: HR CG+GG= 0.66, 95% CI = 0.50 – 0.87, p = 0.0037; OS, modello dominante = HR CG+GG= 0.66, 95% CI = 0.46 – 0.95, p = 0.023, secondo il test dei ranghi logaritmici). Questo significa che pazienti portatori di almeno un allele G non solo hanno dimostrato una miglior risposta clinica al trattamento ma hanno anche avuto una progressione più tardiva ed una sopravvivenza più lunga dei pazienti wild type. Per quanto riguarda il gene dell’aromatasi, l’unica associazione riscontrata, anche se marginale, riguarda il polimorfismo CYP19A1_1558T/C (rs10046) il cui allele variante C che è stato associato ad un ridotto TTP (HRCC modello recessivo =1.4, 95%CI = 1.04 – 1.89, p = 0.028, secondo il Test Esatto di Fischer a due vie). Al contrario, non è stata riscontrata alcuna associazione significativa tra lo SNP CYP19A1_Ex11_410A/C (rs4646), obiettivo principale dello studio, e RR, CB, TTP o OS. Riguardo i polimorfismi del gene dell’aromatasi, siamo stati in grado di descrivere una nuova variante genetica per il polimorfismo STR CYP19A1_(TTTA)n (rs60271534) dell’introne 4. Le banche dati genetiche e la letteratura riportano che il numero di ripetizioni della quadripletta TTTA vari tra 7 e 13, ma nel nostro studio è stato individuato un allele, finora mai descritto, con 14 ripetizioni. Conclusioni: in conclusione, questo lavoro di tesi ha permesso di definire un nuovo biomarcatore molecolare, lo SNP CYP1B1*3_4326G/C (rs1056836), con un valore predittivo e prognostico per il trattamento a base di exemestane in pazienti affetti da ER+ BC, metastatico o localmente avanzato. Questo presuppone che, se validato, questo biomarcatore potrebbe potenzialmente essere impiegato nella pratica clinica oncologica quotidiana come strumento che potrebbe aiutare ad identificare i pazienti che hanno una maggiore probabilità di risposta all’exemestane tramite una semplice valutazione genetica da sangue periferico da effettuarsi prima della terapia. Inoltre, è stata descritta una nuova variante genetica del gene dell’aromatasi

Pharmacogenetics Biomarkers and Their Specific Role in Neoadjuvant Chemoradiotherapy Treatments: An Exploratory Study on Rectal Cancer Patients

Background: Pathological complete response (pCR) to neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) is still ascribed to a minority of patients. A pathway based-approach could highlight the predictive role of germline single nucleotide polymorphisms (SNPs). The primary aim of this study was to define new predictive biomarkers considering treatment specificities. Secondary aim was to determine new potential predictive biomarkers independent from radiotherapy (RT) dosage and cotreatment with oxaliplatin. Methods: Thirty germ-line SNPs in twenty-one genes were selected according to a pathway-based approach. Genetic analyses were performed on 280 LARC patients who underwent fluoropyrimidine-based CRT. The potential predictive role of these SNPs in determining pathological tumor response was tested in Group 1 (94 patients undergoing also oxaliplatin), Group 2 (73 patients treated with high RT dosage), Group 3 (113 patients treated with standard RT dosage), and in the pooled population (280 patients). Results: Nine new predictive biomarkers were identified in the three groups. The most promising one was rs3136228-MSH6 (p = 0.004) arising from Group 3. In the pooled population, rs1801133-MTHFR showed only a trend (p = 0.073). Conclusion: This exploratory study highlighted new potential predictive biomarkers of neoadjuvant CRT and underlined the importance to strictly define treatment peculiarities in pharmacogenetic analyses

New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III-IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor