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

Human RSPO1/R-spondin1 Is Expressed during Early Ovary Development and Augments β-Catenin Signaling

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6–9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination

Pharmacogenetics of cytochromes in breast cancer treatment

Pharmacogenetic focuses on intersubject variations in therapeutic drug effects and toxicity depending on genetic polymorphisms. In defining the treatment strategies for breast cancer several factors are considered: patients with estrogen-positive breast cancer are treated with anti-estrogens (exemestane) whereas locally advanced or metastatic disease is treated with cytotoxic agents, including cyclophosphamide. In both cases, the presence of genetic alterations of cytochrome P450 (CYP) could influence the absorption, distribution, bioactivation, metabolism, elimination and drug action and thus have consequences in the efficacy and toxicity of treatment. This study is focused on the analysis of genetic polymorphisms of several CYP isoforms involved in cyclophosphamide bioactivation or as exemestane target. The aim of this research is to point out a predictive role of genetic alterations in terms of toxicity and of overall survival after pharmacological treatment in breast cancer patients. Since the difficulty of the enrollment of patients treated with exemestane did not allow us to drawn the same pharmacogenetic/pharmacodynamic correlations. For this subgroup we have assessed only the relative risk to develop breast cancer for all the polymorphisms analysed. One hundred ninty-five patients treated with cyclophosphamide in association with methotrexate and 5-fluorouracil (CMF regimen) were genotypized for a set of genetic polymorphisms of CYP2B6, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 involved in bioactivation of the drug. In the group of 121 patients treated with exemestane the analysis were performed for aromatase (CYP19) gene, which is also the target of the drug. Genotyping was performed using PCR based methods, such as Restriction Fragment Length Polymorphism (RFLP), fragment analysis, Pyrosequencing technology and TaqMan technology. Clinical parameters of toxicity (according with NCI-CTC scale) and Overall Survival were monitored by an oncologist. Interesting results were obtained for the correlations between the genotypic and the pharmacodynamic profiles of subgroup of patients treated with cyclophosphamide. The study underlined the significant role of CYP2C9 isoforms involved in bioactivation of cyclophosphamide both in overall survival and in the development of toxicity. Patients carrying at least one mutant allele of CYP2C9*2 (C430T) polymorphism showed an higher risk to develop hepatic toxicity with respect to wild type patients after the 1st cycle of chemotherapy. Similar results were obtained with CYP2C9*3 (A1075C): the presence of at least one mutated allele was found significantly correlated with the development of hepatic toxicity (grade 1-3) after the entire course of treatment. A reduction of enzyme activity due by polymorphism could determine an impaired bioactivation of the prodrug with a consequent accumulation in the liver. Moreover, CYP2C9*3 polymorphism has showed an impact in the overall survival: the presence of at least one mutated allele is related to a reduced survival. This is probably due to a lesser bioavailability of the active molecule. The presence of CYP2C19 G681A polymorphism was correlated with an higher risk to develop severe (grade 3-4) hematological toxicity at the end of the therapy. This variant characterizes the poor metaboliser (PM) allele and thus could be responsible for a reduction in the bioactivation of cyclophosphamide. In conclusion, interesting molecular markers with a predictive value on pharmacodynamics of cyclophosphamide were identified, but their role has to be confirmed in a larger group of patients. A possible application of these parameters in the clinical practice, could be useful to design a tailored treatment based on the peculiar genetic characteristics of each breast cancer patient.La farmacogenetica studia l’impatto di polimorfismi genetici nella variabilità interindividuale dell’effetto terapeutico, in termini di risposta e tossicità, al trattamento farmacologico. Nella definizione del trattamento per il tumore della mammella si tiene conto di numerosi fattori tra cui la condizione ormonale delle pazienti stesse: la presenza di recettori per gli estrogeni a livello tumorale prevede il trattamento con farmaci anti-estrogenici (exemestane) mentre la malattia localmente avanzata o metastatica viene trattata con chemioterapia citotossica (ciclofosfamide). In entrambi i casi, la presenza di alterazioni genetiche a carico dei citocromi P450 (CYP) potrebbe influenzare la terapia in termini di risposta o di sviluppo di tossicità. Lo studio ha previsto l’arruolamento di due gruppi di pazienti sottoposte a terapia farmacologica (ciclofosfamide in regime CMF) o a terapia ormonale (exemestane) per le quali è stato fatto il profilo genetico di alcune particolari isoforme di CYP. Lo scopo dello studio è stato quello di valutare l’effetto dei polimorfismi individuati sull’outcome clinico delle pazienti in modo da fornire delle indicazioni su una possibile “personalizzazione” della terapia in relazione al profilo genetico della paziente. Lo studio si può definire completo per il gruppo di pazienti in trattamento farmacologico in quanto è stato possibile raccogliere i dati farmacodinamici di tossicità e sopravvivenza. Al contrario, per il gruppo in trattamento ormonale l’arruolamento non è stato completato quindi è stato possibile solo avere dei dati preliminari di frequenza dei polimorfismi. Lo scopo principale dello studio è stato quindi quello di valutare l’impatto di polimorfismi a carico dei CYP (siano essi coinvolti nel metabolismo della ciclofosfamide o rappresentino l’enzima target per exemestane) sulla sopravvivenza e sullo sviluppo di tossicità al trattamento. Inoltre, è stato possibile effettuare anche delle correlazioni di tipo caso/controllo per la valutazione del rischio relativo di sviluppo del tumore in relazione alla presenza dei polimorfismi studiati. Sono state quindi individuate le varianti genetiche a carico delle isoforme CYP2B6, CYP2C9, CYP2C19, CYP3A4 e CYP3A5 coinvolte nella bioattivazione della ciclofosfamide e dell’isoforma CYP19 enzima target di exemestane. La genotipizzazione delle pazienti (195 con trattamento farmacologico e 121 con trattamento anti-estrogenico) si è basata su tecniche che utilizzano l’amplificazione del DNA tramite PCR, quali il Restriction Fragment Length Polymorphism (RFLP), l’analisi automatizzata dei frammenti, la tecnologia del Pyrosequencing e la tecnologia TaqMan. I dati clinici di tossicità sono stati valutati da un oncologo medico secondo la classificazione NCI-CTC mentre il dato di sopravvivenza è stato considerato l’Overall Survival. Dati interessanti e significativi sono emersi per il gruppo di pazienti in trattamento farmacologico con ciclofosfamide in quanto è stato possibile valutare le relazioni tra profilo genetico e profilo farmacodinamico. Lo studio ha infatti evidenziato un ruolo significativo dei polimorfismi dell’isoforma CYP2C9 coinvolti nella bioattivazione epatica della ciclofosfamide sia per quanto riguarda la sopravvivenza che la tossicità. La presenza di almeno un allele mutato per il polimorfismo CYP2C9*2 (C430T) è correlata con un maggior rischio di sviluppo di tossicità epatica alla fine del primo ciclo di trattamento. La riduzione di attività enzimatica conseguente alla variazione genica potrebbe determinare una minore bioattivazione del profarmaco con un suo accumulo a livello epatico. Inoltre anche il polimorfismo CYP2C9*3 (A1075C) ha evidenziato un impatto non solo sulla tossicità epatica ma anche sulla sopravvivenza. La presenza di almeno un allele mutato riduce la sopravvivenza probabilmente in relazione alla minore disponibilità di farmaco attivo. Inoltre è emersa una correlazione significativa anche tra la presenza di un polimorfismo a carico di CYP2C19 (G681A) e lo sviluppo di tossicità ematologica severa (grado 3-4) alla fine della terapia. La presenza del polimorfismo è associata ad una riduzione nell’attività metabolica dell’enzima quindi potrebbe spiegare una riduzione nella bioattivazione della ciclofosfamide con sviluppo di tossicità. In conclusione, durante questo lavoro di tesi, sono stati evidenziati interessanti marcatori molecolari che potrebbero avere un valore prognostico nel trattamento del carcinoma della mammella con ciclofosfamide se confermati in una casistica più ampia. L’applicazione di questi parametri nella pratica clinica potrebbe essere utile per progettare una personalizzazione del trattamento basato su specifiche caratteristiche genetiche del paziente con carcinoma della mammella

Candidate germline polymorphisms of genes belonging to the pathways of four drugs used in osteosarcoma standard chemotherapy associated with risk, survival and toxicity in non-metastatic high-grade osteosarcoma

This study aimed to identify associations between germline polymorphisms and risk of high-grade osteosarcoma (HGOS) development, event-free survival (EFS) and toxicity in HGOS patients treated with neo-adjuvant chemotherapy and surgery. Germline polymorphisms of 31 genes known to be relevant for transport or metabolism of all four drugs used in HGOS chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) were genotyped in 196 patients with HGOS and in 470 healthy age and gender-matched controls. Of these 196 HGOS patients, a homogeneously treated group of 126 patients was considered for survival analyses (survival cohort). For 57 of these, treatment-related toxicity data were available (toxicity cohort). Eleven polymorphisms were associated with increased risk of developing HGOS (p < 0.05). The distribution of polymorphisms in patients was characterized by a higher Shannon entropy. In the survival cohort (n = 126, median follow-up = 126 months), genotypes of ABCC2_1249A/G, GGH_452T/C, TP53_IVS2+38G/C and CYP2B6*6 were associated with EFS (p < 0.05). In the toxicity cohort (n = 57), genotypes of ABCB1_1236T/C, ABCC2_1249A/G, ABCC2_3972A/G, ERCC1_8092T/G, XPD_23591A/G, XRCC3_18067T/C, MTHFR_1298A/C and GGH_16T/C were associated with elevated risk for toxicity development (p < 0.05). The results obtained in this retrospective study indicate that the aforementioned germline polymorphisms significantly impact on the risk of HGOS development, EFS and the occurrence of chemotherapy-related toxicity. These findings should be prospectively validated with the aim of optimizing and tailoring HGOS treatment in the near future

Prognostic Role of a New Index (RAPID Index) in Advanced Hepatocellular Carcinoma Patients Receiving Sorafenib: Training and Validation Cohort

Background and Aims: The aim of the present study is to evaluate a new index influenced by the balance between the immune system, α-fetoprotein (AFP), and lactate dehydrogenase (LDH) (RAPID index) as a prognostic factor in patients treated with sorafenib. Methods: This study was conducted on a training cohort of 159 hepatocellular carcinoma (HCC) patients and a validation cohort of 68 HCC patients treated with sorafenib. The RAPID index was calculated as neutrophil/lymphocyte count × LDH × AFP. Results: In the training cohort, the median overall survival (OS) was 23.2 months (95% CI 11–25) and 12.1 months (95% CI 9–15) for patients with a low (≤3,226) and high (&gt;3,226) RAPID index, respectively (ref. &lt;3,226, HR = 0.56, 95% CI 0.35–0.88, p = 0.017). Following adjustment for clinical covariates, multivariate analysis confirmed the RAPID index ≤3,226 versus &gt;3,226 (HR = 0.37, 95% CI 0.18–0.74, p = 0.0054) as an independent prognostic factor for OS. In the validation cohort, the median OS was 26.9 months (95% CI 17.6–26.9) and 7.0 months (95% CI 6.2–9.2) for patients with a low (≤ 3,226) and high (&gt;3,226) RAPID index, respectively (ref. &lt;3,226, HR = 0.19, 95% CI 0.10–0.36, p &lt; 0.0001). Performing the same multivariate analysis of the training cohort (AFP, Eastern Cooperative Oncology Group, aspartate aminotransferase, neutrophil, platelet, systemic inflammatory index and RAPID index), the RAPID index &lt;3,226 versus &gt;3,226 (HR = 3.86, 95% CI 1.45–10.29, p = 0.007) was found to be an independent prognostic factor for predicting OS. Conclusion: The low cost, easy assessment, and reproducibility of a full blood count make the RAPID index a promising tool for assessing HCC prognosis in future clinical practice

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases

More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD)