Individual variations in motives for nicotine self-administration in male rats: evidence in support for a precision psychopharmacology.

The significant heterogeneity in smoking behavior among smokers, coupled with the inconsistent efficacy of approved smoking cessation therapies, supports the presence of individual variations in the mechanisms underlying smoking. This emphasizes the need to shift from standardized to personalized smoking cessation therapies. However, informed precision medicine demands precision fundamental research. Tobacco smoking is influenced and sustained by diverse psychopharmacological interactions between nicotine and environmental stimuli. In the classical experimental rodent model for studying tobacco dependence, namely intravenous self-administration of nicotine, seeking behavior is reinforced by the combined delivery of nicotine and a discrete cue (nicotine+cue). Whether self-administration behavior is driven by the same psychopharmacological mechanisms across individual rats remains unknown and unexplored. To address this, we employed behavioral pharmacology and unbiased cluster analysis to investigate individual differences in the mechanisms supporting classical intravenous nicotine self-administration (0.04 mg/kg/infusion) in male outbred Sprague-Dawley rats. Our analysis identified two clusters: one subset of rats sought nicotine primarily for its reinforcing effects, while the second subset sought nicotine to enhance the reinforcing effects of the discrete cue. Varenicline (1 mg/kg i.p.) reduced seeking behavior in the former group, whereas it tended to increase in the latter group. Crucially, despite this fundamental qualitative difference revealed by behavioral manipulation, the two clusters exhibited quantitatively identical nicotine+cue self-administration behavior. The traditional application of rodent models to study the reinforcing and addictive effects of nicotine may mask individual variability in the underlying motivational mechanisms. Accounting for this variability could significantly enhance the predictive validity of translational research.

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

Identification of a novel large EPCAM-MSH2 duplication, concurrently with LOHs in chromosome 20 and X, in a family with Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is associated with germline mutations in one of the mismatch repair genes or EPCAM. The majority of the causative alterations are point mutations. Large genomic rearrangements represent only 5-20%. Hypothetically, the allelic imbalance, like the loss of heterozygosity, may be another high penetrance risk factor. CASE PRESENTATION: We describe the case of a patient who developed 5 tumors during her lifetime and with a family history characterized by a high frequency of tumors associated with LS. The proband was tested for mutations and copy number alterations with a panel of hereditary cancer genes and by SNP array. She showed a 187 Kb duplication including EPCAM and the first 7 exons of MSH2, plus two loss of heterozygosity (LOHs) in chromosome 20 and one in chromosome X which include many tumor suppressor genes. CONCLUSION: We found a novel large EPCAM-MSH2 duplication associated with LS and the presence of LOHs in regions containing numerous tumor suppressors, raising the hypothesis that these alterations could contribute to cancer susceptibility. Our results underline the importance to deepen the knowledge of molecular mechanisms in order to determine the role in cancer predisposition of novel genetic alterations.

Heterogeneity in Colorectal Cancer: A Challenge for Personalized Medicine?

High inter-patient variability and high spatial heterogeneity are features of colorectal cancer (CRC). This may influence the molecular characterization of tumor tissue, now mandatory for patients with metastatic CRC who are candidates for treatment with an anti-EGFR mAb, as false-negative results can occur, leading to non optimal therapy. Moreover, temporal molecular heterogeneity during treatment is known to influence the response to therapy and prognosis. We present a literature overview of advances made in characterizing molecular heterogeneity in CRC, underlining that the analysis of liquid biopsy could represent an efficient non-invasive tool to overcome the problem. We believe that understanding CRC heterogeneity is fundamental for a more accurate diagnosis, for selecting the best targets to ensure prolonged antitumor response, and for monitoring minimal residual disease and the onset of resistance to therapy, all essential components of successful personalized treatment.

Shifts of Faecal Microbiota During Sporadic Colorectal Carcinogenesis.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.

Stool DNA Integrity Method for Colorectal Cancer Detection.

Fluorescence long DNA (FL-DNA) is a non-invasive and simple-to-perform stool DNA test. This assay consists of a qualitative and quantitative real-time PCR (RT PCR) analysis. FL-DNA has great potential in colorectal cancer (CRC) lesions detection used alone or in combination with the standard CRC screening tool: immunochemical fecal occult blood test (iFOBT).

What influences preneoplastic colorectal lesion recurrence?

The hypothesis of the local recurrence of preneoplastic lesions was first put forward in the 1950s. Disease recurrence may result from an inherent imbalance in cell proliferation that promotes carcinogenesis in apparently normal mucosa. Our review sheds light on how early preneoplastic lesions could be used to diagnose relapsed preneoplastic and, developing neoplastic lesions. We focus in detail on the clinical-pathological and molecular features of adenoma subtypes and their role in relapsed adenoma and their development into colorectal carcinoma. Moreover, we include the data available on microbiota and its metabolites and their role in recurrence. We strongly believe that a significant improvement could be achieved in colorectal screening by introducing personalized endoscopic surveillance for polyp-bearing patients on the basis of the presence of molecular markers that are predictive of recurrence.

Promoter methylation of tumor suppressor genes in pre-neoplastic lesions; potential marker of disease recurrence.

BACKGROUND: Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence. METHODS: A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry. RESULTS: Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively. CONCLUSIONS: Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

Improved stool DNA integrity method for early colorectal cancer diagnosis.

BACKGROUND: DNA integrity analysis could represent an alternative approach to the early detection of colorectal cancer. Previously, fluorescence long DNA (FL-DNA) in stools was extracted using a manual approach and analyzed by capillary electrophoresis assay (CE FL-DNA). We aimed to improve diagnostic accuracy using a simpler and more standardized method [Real Time PCR FL-DNA (RT FL-DNA)] for the detection of early malignant lesions in a population undergoing colorectal cancer screening. METHODS: From 241 stool samples, DNA was extracted using manual and semiautomatic extraction systems and analyzed using FL-DNA tests by CE and RT assays. The RT FL-DNA approach showed slightly higher sensitivity and specificity compared with the CE FL-DNA method. Furthermore, we compared the RT FL-DNA approach with the iFOBT report. RESULTS: Nonparametric ranking statistics were used to analyze the relationship between the median values of RT FL-DNA and the clinicohistopathologic characteristics. The median values of both variables were significantly higher in patients with cancer than in patients with noncancerous lesions. According to the Fagan nomogram results, the iFOBT and FL-DNA methods provided more accurate diagnostic information and were able to identify subgroups at varying risks of cancer. CONCLUSIONS: The combination of the semiautomatic extraction system and RT FL-DNA analysis improved the quality of DNA extracted from stool samples. IMPACT: RT FL-DNA shows great potential for colorectal cancer diagnosis as it is a reliable and relatively easy analysis to perform on routinely processed stool samples in combination with iFOBT.

Circulating and stool nucleic acid analysis for colorectal cancer diagnosis.

In recent years, the need to identify molecular markers characterized by high sensitivity and specificity in detecting and monitoring early and colorectal cancer lesions has increased. Up to now, none of the markers or panels of markers analyzed have met the rigorous standards required of a screening program. The important discovery of circulating nucleic acids in biological fluids has aroused intense scientific interest because of their usefulness in malignant and non malignant diseases. Over time, their yield and stability have been identified and compared with other "standard" biomarkers. The analysis of circulating DNA from blood and stool is a relatively simple and non-invasive procedure, representing a very attractive marker to detect genetic and epigenetic mutations and to monitor disease progression. A correlation between blood and stool biomarkers could also help to enhance currently available diagnostic approaches. However, various processing and analytic problems need to be resolved before such an approach can be applied in clinical practice.