Ermanno Nardon

Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation.

Formalin-fixed tissues represent the most abundant clinical material for retrospective studies. However, formalin highly affects macromolecules, impairing their extraction and analysis. In this study, the suitability of some potential substitutes of formalin for RNA-based applications has been considered. Conventional formalin was compared with methacarn and the commercial FineFIX. Their impact on overall RNA preservation was investigated in a cell line-based model fixed during a time course treatment and in a series of fixed human tissues. RNA yield was detected by Nanodrop; ribosomal RNA (rRNA) integrity by electrophoresis and the Agilent Bioanalyzer; messenger RNA (mRNA) integrity by Northern blot and endpoint reverse transcription-polymerase chain reaction; and mRNA amount by real-time polymerase chain reaction. In the cell line model, formalin fixation showed time-dependent detrimental effects on overall RNA preservation. Methacarn and FineFIX were more conservative on both rRNA and mRNA preservation and their impact was time-independent. In tissues, high rRNA degradation levels were found in all fixed specimens, contrasting with the results found in the cells. Conversely, the effects of the fixatives on mRNA integrity reflected the observations shown in the cell line model. In methacarn-fixed samples mRNA amount was also preserved, whereas in formalin and FineFIX-fixed samples it was notably lower when compared with the fresh frozen control. Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation. In fixed tissue samples, however, the potential effects of preanalytical tissue-related factors should be considered when performing mRNA quantitative analysis.

Prevalence of Different Subtypes of Serrated Polyps and Risk of Synchronous Advanced Colorectal Neoplasia in Average-Risk Population Undergoing First-Time Colonoscopy

OBJECTIVES:A growing body of evidence indicates that patients with sessile serrated adenoma/polyp (SSA/P) and traditional serrated adenoma (TSA) are at risk for subsequent malignancy. Despite increasing knowledge on histological categorization of serrated polyps (SPs) data are lacking on the actual prevalence and the association of each SP subtype with advanced colorectal neoplasia.METHODS:We prospectively determined the prevalence of different SP subtypes and evaluate the association with synchronous advanced neoplasia in asymptomatic average-risk subjects undergoing first-time colonoscopy. All retrieved polyps were examined by two independent pathologists. Serrated lesions were classified into hyperplastic polyps (HP), SSA/P (without and with cytological dysplasia, SSA/P/DIS), and TSA, and were screened for BRAF and K-ras mutations.RESULTS:Among 258 polyps detected in 985 subjects, the proportion of SSA/P and TSA was 8.9% and 1.9% with an overall prevalence of 2.3% and 0.6%, respectively. SSA/Ps were small without significant difference in their location between proximal and distal colon; TSA were predominantly left-sided. BRAF mutation was common in SSA/Ps and K-ras mutation was present in all TSA. Independent predictors of advanced neoplasia were male sex (odds ratio (OR)=2.0, 95% confidence interval (CI) 1.0–4.0), increasing age (OR=4.5, 95% CI 1.5–13.4 for 50–69 years and OR=9.9, 95% CI 3.1–31.5 for >70 years), current smoking (OR=2.0, 95% CI 1.3–6.8), >3 tubular adenoma (OR=3.6, 95% CI 1.9–6.4), and SSA/P (OR=6.0, 95% CI 1.9–19.5).CONCLUSIONS:The substantial prevalence of BRAF-mutated SSA/P and the independent association with synchronous advanced colorectal neoplasia in asymptomatic average-risk subjects support the overall impact of the serrated pathway on colorectal cancer (CRC) risk in general population. The endoscopic characteristics of SSA/P emphasize the need of high-quality colonoscopy as a key factor for an effective CRC screening program.

A Multicenter Study to Validate the Reproducibility of MSI Testing With a Panel of 5 Quasimonomorphic Mononucleotide Repeats.

Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.

Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability.

Real time quantitative reverse transcription-PCR (qRT-PCR) is the most sensitive technique for detection and quantification of mRNA targets. Reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissues (FFPE), however, has been subjected to serious limitations so far, mainly due to the fragmentation of RNA transcripts. We tried to improve the sensitivity and reliability of mRNA quantification in FFPE by boosting the reverse transcription (RT) step, that is neglected in most of the protocol analysis, but that represents the first confounding event in a quantitative analysis. For this purpose, we compared yield, reproducibility and linearity of RTs performed with random hexamers, random pentadecamers, or a mixture of antisense specific primers in presence of either Moloney murine leukemia virus (MmLV) or the avian myeloblastosis virus (AMV) enzymes. Random primers were tested at two concentrations, 0.14 and 3.35 nmol/reaction. Our qRT-PCR results indicate an improvement of RT yield when using the highest concentration of random oligos with MmLV (from -1.4 to -4.1 C(t)s) in comparison to the lowest concentration. Moreover, more reliable standard curves and therefore, efficiencies were obtained. RT reactions performed with specific primers and AMV were those with the highest yield, but efficiencies were unreliable, due to the RT enzyme-driven PCR inhibition. Random priming at the 3.35 nmol/reaction concentration seems to be the most convenient strategy in assays using RNA obtained from FFPE tissues.

Quantitative Real-Time RT-PCR

Quantitative real-time PCR is a sensitive tool for quantitative expression analysis of RNA isolated from clinical samples and is increasingly being utilized in novel clinical diagnostic assays. It is an essential tool for the detection of degraded RNA, as that extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Although it has unquestionable potential for expression analysis in archival case studies, there are still some biological and technical problems associated with its use, which can be partly overcome by a standardization of the protocols. The first part of this chapter provides a detailed description of the most commonly used real-time-based chemistries, SybrGreen and TaqMan, focusing especially on their advantages when applied in FFPE material. Specific suggestions have been included in order to improve the reliability of the expression results. The second part of the chapter is dedicated to the description of general guidelines that should be followed to optimize the design of quantitative real-time PCR assays. They have been adapted to RNA isolated from FFPE but should always be taken into account in any gene expression analysis to ensure its accuracy and reproducibility.

Scarce information about the risk of cancer in colorectal hyperplastic polyps and polyposis

Scarce information about the risk of cancer in colorectal hyperplastic polyps and polyposis

Qualitative Methylation Status Assessment

Methylation of cytosines in CpG dinucleotides is an important regulator of gene expression in the human genome. Aberrant methylation is frequently observed in carcinogenesis and its detection has been exploited for diagnostic purposes, for prognosis, or as a predictive tool of response to therapy. Currently, most methods for assessing methylation rely on bisulfite conversion of the DNA, which causes cytosine conversion to uracil, while leaving methylated cytosines unmodified. Since this modification is detrimental for DNA quality and could yield artifacts due to incomplete conversion, its chemistry needs to be tailored to the analyzed material. This chapter provides the protocols for: a single-locus assay relying on a nested, bisulfite modification-specific methylation-specific PCR (BM-MSP) design. protocols for bisulfite modification and DNA extraction consistent with FFPE-derived material are also supplied. a multilocus assay based on methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), which is particularly suitable for FFPE as it does not need bisulfite conversion and performs well on short sequences.

Quantitative Methylation Status Assessment in DNA from FFPE Tissues with Bisulfite Modification and Real-Time Quantitative MSP

The quantitative assessment of methylation at specific sequences can be requested whenever a pathological condition can be identified or stratified if it exceeds a given methylation threshold level. Real-time qPCR technology can be consistently exploited for detecting the presence and the amount of specific methylation patterns in sequences of interest, after bisulfite conversion of DNA (Methylight). This chapter provides a protocol for assessing the methylation status of five surrogate markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) for the presence of the CpG Island Methylator Phenotype (CIMP) in human colorectal cancer samples. Methylation level at each locus is expressed as a percentage relative to a fully-methylated, M.SssI-treated reference DNA sample (Percentage of Methylated Reference, PMR). Each methylation datum is normalized against the amount of fully converted DNA, assessed with a control reaction targeting the repetitive element ALU-C4. A sample is defined as CIMP+ if showing a PMR>;10 in at least three of the five markers. This chapter also provides the methods for M.SssI and Bisulfite modifications most suitable for use with Methylight technology in FFPE samples.

Standards for Equipment and Reagents

The proper maintenance of laboratory equipment and the need for high-quality reagents were earlier recognized as core elements of Good Laboratory Practice (GLP) from international regulatory agencies. Most of the equipment and of the reagents used in molecular pathology laboratories were designed for research and successively adapted for clinical use. For these reasons, the instrumentation requires rigorous surveillance of its performance and prompt technical support; chemicals need proper storage and periodical monitoring of their quality. This chapter focuses on a collection of GLP-compliant suggestions for proper management of the basic laboratory equipment and reagents. Although relevant for an assay performance, these items are often neglected and researchers are faced with unexpected assay results or unexplainable test failures: laboratory compliance to GLP principles is indubitably demanding but allows getting rid of many confounder elements. The standards described in this chapter are based partly on applicable parts of existing guidance elements (in particular, the Laboratory Requirements as described by 42 CFR part 493 of CLIA) and partly on sound lab practices followed in the IMPACTS institutions.

Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues

Microsatellite sequences are genome-wide dispersed stretches of short tandem nucleotide repeats. Because of their repetitive nature, microsatellites are prone to undergo shortening or extension during DNA replication because of polymerase slippage or misalignment of template strands (microsatellite instability, MSI). As spontaneous mutation rate increases dramatically in the presence of a defective mismatch repair (MMR) system, MSI represents an ideal phenotypic indicator of an MMR defect. MSI occurs in approximately 15% of colorectal cancers, including those arising in the Hereditary Non-Polyposis CRC familiar syndrome (HNPCC or Lynch Syndrome). MSI tumours feature a series of molecular and clinicopathological signatures that are distinct from non-MSI ones. MSI testing therefore enables identification of patients having a unique prognosis and a different response to particular drug therapies. This chapter provides three methodological approaches for assessing MSI in FFPE samples: a basic method involving amplification of the NCI-validated microsatellite marker sequences, with PAGE run and silver stain detection of PCR products; a multiplex PCR amplification of five mononucleotide markers alternative to the NCI panel, coupled with DHPLC (Denaturating High-Performance Liquid Chromatography) analysis of PCR products; and a multiplex PCR amplification of the five mononucleotide markers coupled with capillary electrophoresis.