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Supplementary Figure 1. Fagan Nomogram analysis. A line is drawn connecting the pre-test probability and the point on the middle vertical line corresponding to the likelihood ratio for the test result, represented by a range of test results (boxes). This line is extended to intersect with the right-hand vertical line, which gives the post-test probability. This point is the new estimate of probability that the patient has the disease. A) Nomogram for post-test probability of having a tumor. B) Nomogram for post-test probability of having a tumor or high-risk adenoma.

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. Cancer Epidemiol Biomarkers Prev; 23(11); 2553–60. ©2014 AACR.

Supplementary Table 1. CRC and CRC+HRA prevalence as a function of different FL-DNA cut offs in negative-positive iFOBTs separated by the median value of all positive iFOBTs

Diet is a major driver of gut microbiota variation and plays a role in metabolic disorders, including metabolic syndrome (MS). Mycorrhized foods from symbiotic agriculture (SA) exhibit improved nutritional properties, but potential benefits have never been investigated in humans. We conducted a pilot interventional study on 60 adults with ≥ 1 risk factors for MS, of whom 33 consumed SA-derived fresh foods and 27 received probiotics over 30 days, with a 15-day follow-up. Stool, urine and blood were collected over time to explore changes in gut microbiota, metabolome, and biochemical, inflammatory and immunologic parameters, previous dietary habits were investigated through a validated food-frequency questionnaire. The baseline microbiota showed alterations typical of metabolic disorders, mainly an increase in Coriobacteriaceae and a decrease in health-associated taxa, which were partly reversed after the SA-based diet. Improvements were observed in metabolome, MS presence (two out of six subjects no longer had MS) or components. Changes were more pronounced with less healthy baseline diets. Probiotics had a marginal, not entirely favorable, effect, although one out of three subjects no longer suffered from MS. These findings suggest that improved dietary patterns can modulate the host microbiota and metabolome, counteracting the risk of developing MS.

// Giulia De Maio 1 , Elisa Zama 1 , Claudia Rengucci 1 , Daniele Calistri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC), Italy Correspondence to: Daniele Calistri, email: daniele.calistri@irst.emr.it Keywords: recurrence, preneoplastic lesions, suppressor and serrated pathway, microbiota Received: August 02, 2016 Accepted: November 15, 2016 Published: November 25, 2016 ABSTRACT 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.

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.

Background: The origin and mechanisms of chromosomal instability are still widely unknown. We previously investigated a limited number of human sporadic colorectal cancers (CRCs) and observed a statistically different occurrence of KRAS and p53 mutations among predetermined subgroups of tumors with different degrees of DNA aneuploidy. The aim of the present study was to further verify these observations by including BRAF gene analysis and by investigating a larger series of cases subdivided into Dukes' stages A to D to reconstruct some form of chronological modulation for events during CRC progression. Methods: KRAS, p53, BRAF mutations and flow cytometric DNA Index were evaluated by established techniques in a series of 135 human sporadic CRCs. Results: p53, KRAS and BRAF mutations were found in 39%, 34%, and 4% of tumors, respectively. The frequency of p53 mutations increased from 15% for stage A to 48% for stage D and was highest in near-diploid (DI 1.6) tumors. A similar correlation between gene mutations and DI values was observed for KRAS. The simultaneous presence of KRAS and p53 mutations was observed in only 11% of cases. Moreover, the co-occurrence of p53 and KRAS mutations was only observed in near-diploid and high-aneuploid tumors. Conclusion: Our findings suggest that KRAS and p53 gene mutations, which are rarely simultaneous and are associated with specific DI aneuploid values, do not represent a synergistic evolutionary pathway but may influence mechanisms of chromosomal instability.

Background.Genomic studies have shown that gene expression profiles are similar inin situ(CIS) and invasive breast cancers, suggesting that several biofunctional modifications of the transformation process occur before or during the development of CIS lesion.Methods.We investigated 3 biomarkers in 44 patients with CIS: TG2 (transglutaminase 2), HJURP (Holliday junction recognition protein), and HIF-1α (hypoxia inducible factor-1 alpha).Results.TG2 was more highly expressed than the other two markers and significantly more so in stromal than in tumor cells. HIF-1α evaluation showed a higher expression in both tumor and stromal cells in patients with relapsed G3 tumors, indicating a potential role of this marker in CIS evolution. A greater than sevenfold higher risk of relapse(P=0.050)was observed in patients highly expressing HJURP in stroma and a tenfold higher recurrence risk(P=0.026)was seen in those with a higher stromal HIF-1α expression. An important increase in risk accuracy (AUC 0.80) was obtained when HIF-1α and HJURP were evaluated together.Conclusions.Despite the limited number of relapsed patients, we formulated some hypotheses on the factors responsible for malignant evolution and recurrence which are now being tested in a large case series with a longer follow-up.

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.

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. Cancer Epidemiol Biomarkers Prev; 23(11); 2553–60. ©2014 AACR.

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.

We previously described a cohort of grade II oligodendroglioma (OII) patients, in whom the loss of heterozygosity (LOH) 19q was present in the subgroup at a higher risk of relapse. In this study, we evaluated the CpG methylation of the putative tumor suppressor epithelial membrane protein 3 (EMP3, 19q13.3) gene promoter in the same OII cohort, to investigate whether a correlation could be found between EMP3 cytogenetic and epigenetic loss and higher risk of relapse. Twenty-three tumor samples from OII patients were collected over a period of 10 years. Seventeen glioblastoma (GBM) samples (2 of which were relapses) were collected from 15 patients. The EMP3, O6-methylguanine methyltransferase (MGMT) and cyclooxygenase 2 (COX2) promoter methylation, evaluated by methylation-specific PCR, and the isocitrate dehydrogenase 1 (IDH1) mutation, identified by sequencing, were compared between the OII and GBM histotypes. The EMP3 promoter methylation was correlated with the analysis of LOH 19q, performed by microsatellite amplification, in OII patients. Disease progression-free interval was evaluated in the OII patients with the EMP3 methylation with either LOH 19q or conserved chromosome 19 arms. The EMP3 and MGMT promoter methylation was more frequent in OII than in GBM patients, and the IDH1 mutation was absent in GBM. The COX2 promoter was unmethylated in both histotypes. Both LOH+/- 19q OII patients showed EMP3 hypermethylation. Concomitant LOH 19q and EMP3 gene promoter methylation was observed in the OII patients at a higher risk of relapse. Our results suggest that a total (cytogenetic and epigenetic) functional loss of both EMP3 alleles accounts for the reduced disease progression-free interval in OII patients. Although the small sample size limits the strength of this study, our results support testing this hypothesis in larger cohorts of patients, considering the methylation of the EMP3 gene promoter together with LOH 19q as an indication for treatment with adjuvant therapy ab initio in order to improve the overall survival of OII patients.

Background: We aimed to define the potential of the fecal DNA assay as an alternative or in addition to the currently used immunochemical fecal occult blood test (iFOBT) for the early diagnosis of colorectal cancer. Methods: A total of 560 individuals aged 50 to 69 years with a positive iFOBT were recruited from an Italian FOBT regional screening program. Twenty-six were diagnosed with adenocarcinoma, 264 with high-risk adenoma, and 54 with low-risk adenoma, whereas 216 subjects did not have premalignant or malignant lesions. Fecal DNA integrity was analyzed blindly by the fluorescence long DNA (FL-DNA) test. Results: iFOBT and FL-DNA were largely independent variables (rs = 0.036, P = 0.42), with values ranging from 101 to 5,826 ng/mL and from 0 to 515 ng, respectively. Median values of both variables were significantly higher in cancer patients than in patients with noncancerous lesions or in healthy individuals. Moreover, iFOBT and FL-DNA values were individually associated with a number of pathologic parameters. Sequential use of the diagnostic iFOBT and FL-DNA methods showed that fecal DNA provided more accurate diagnostic information and was able to identify subgroups at different risk of cancer in iFOBT-positive individuals. Conclusions: A combined approach based on FL-DNA and iFOBT evaluation could help to better identify colorectal cancers and to determine a patient's risk of harboring a preneoplastic or neoplastic lesion. Further evaluation in a screening setting is needed to confirm this hypothesis. Impact: Fecal DNA could be a useful tool to better predict cancer risk in FOBT-positive individuals. Cancer Epidemiol Biomarkers Prev; 19(10); 2647–54. ©2010 AACR.

Background & Aims: Evaluation of molecular alterations in fecal DNA is a potential, noninvasive, alternative tool for the detection of colorectal cancer. We analyzed a large panel of molecular alterations involved in tumor transformation and progression to define their single diagnostic contribution in terms of sensitivity, cost, and time required to carry out the different tests. Methods: DNA was analyzed in stool from 38 healthy individuals and in paired stools and primary lesions from 56 patients with colorectal cancer. p53 exons 5–8, K-ras exons 1–2, four fragments of adenomatous polyposis coli (APC) exon 15, and 5 microsatellite loci were analyzed. Moreover, DNA amplification was evaluated for 4 exons of both p53 and APC. Results: K-ras (34%) and p53 (34%) mutations were the most frequent alterations in tumors, followed by microsatellite instability (13%) and APC mutations (13%). The most frequent event in stool was DNA amplification (51%), followed by alterations of K-ras (11%), p53 and microsatellite instability (6%), and APC (2%). K-ras and p53 gene mutations increased the capacity of DNA amplification to detect tumor cells by 8%. Conclusions: K-ras and p53 gene mutations were the most frequent alterations observed in stool from patients with colorectal cancer, but DNA amplification was even more frequent, being present in more than half of patients. If these preliminary results are confirmed in a prospective study on a larger case series, this approach could be used for noninvasive colon cancer diagnosis in screening programs.

DNA amplification of exfoliated cells in stool repre sents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, calle( fluorescence long DNA, was developed and validate( in our laboratory on stool obtained from 86 patient., with primary colorectal cancer and from 62 health individuals. It consists of the amplification of stoo DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of thi new method compared to the conventional approact was analyzed in a subgroup of 94 individuals (51 patients and 38 healthy volunteers). In the presen series, DNA amplification analysis showed a specific ity of 97% and a sensitivity of only 50%. Conversely fluorescence DNA evaluation, using the best cutoff o 25 ng, showed a sensitivity of about 76% and a spec ificity of 93%. Similar sensitivity was observed regard less of Dukes stage, tumor location, and size, thu., also permitting the detection of early-stage tumors The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.