Accurate Molecular Analysis Research Articles

Evaluation of pathogenic variants detected in high homology regions of the PMS2 gene. How effective is long-range PCR?

Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, most laboratories do not clearly report the origin of this molecular finding. The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the PMS2 gene in a Brazilian sample. An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with PMS2. In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Technical evaluation of a novel digital PCR platform for detecting EGFR/KRAS mutations in NSCLC archived plasma specimens

IntroductionKRAS p.G12C hot spot mutations has rapidly modified diagnostic algorithm for lung cancer patients electing Non-Small Cell Lung Cancer (NSCLC) patients to target treatment. As regards, patients that harbor this hallmark showed a clinical benefit in terms of progression-free survival (PFS) and overall survival (OS) in comparison with control group under target treatment. In this scenario, KRAS p.G12C mutation requires optimized testing strategy in diagnostic routine practice. Although the widespread diffusion of NGS platforms, a not negligible percentage of Italian diagnostic institutions adopt singleplex technology (RT-PCR, dPCR) for molecular testing. Here, we aim to technically validate a novel dPCR system (QIAcuity™ Digital PCR System, Qiagen; Hilden, Germany) on a retrospective series of cfDNA samples from previously tested with a custom NGS system. (1,2)Methods: n = 50 liquid biopsy specimens (n = 25 KRAS/EGFR mutated and n = 25 wild type for actionable KRAS/EGFR mutations) from diagnostic routine NSCLC patients previously tested with a custom NGS panel were retrieved from our internal archival. Each sample was tested by adopting n = 5 KRAS and n = 3 EGFR commercially available dPCR assays on QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany); an ultra-deep dPCR walk-away platform that automatizes molecular analysis. Technical sensitivity, technical specificity, and concordance rate between “gold standard” NGS system and QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany) were assessed. ResultsOverall, all specimens were successfully analyzed with dPCR system. In details, 24 out of 25 mutated and 21 out of 24 wild type cases were detected. A technical sensitivity, specificity, and a concordance rate of 96.0 % (24/25), 88.0 % (22/25) and 92.0 % (46/50) were evaluated taking into account a MAF cut-off ≥ 0.2 % and a partition number of 100 positive partitions in wild-type channel. ConclusionQiacuity (Qiagen; Hilden, Germany) platform enables accurate molecular analysis of diagnostic routine specimens. Optimized technical workflow is required to technically implement this platform in diagnostic routine setting.

Partitioning and subsampling statistics in compartment-based quantification methods.

The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.

DNA methylation marker to estimate ovarian cancer cell fraction.

Evaluation of a cancer cell fraction is important for accurate molecular analysis, and pathological analysis is the gold standard for evaluation. Despite the potential convenience, no established molecular markers for evaluation are available. In this study, we aimed to identify ovarian cancer cell fraction markers using DNA methylation highly specific to ovarian cancer cells. Using genome-wide DNA methylation data, we screened candidate marker genes methylated in 30 ovarian cancer FFPE samples and 12 high-grade serous ovarian cancer cell lines, and unmethylated in two female leucocytes and two normal fallopian epithelial cell samples. Methylation levels of two genes, SIM1, and ZNF154, showed high correlation with pathological cancer cell fractions among the 30 ovarian cancer FFPE samples (R = 0.61 for SIM1, 0.71 for ZNF154). For cost-effective analysis of FFPE samples, pyrosequencing primers were designed, and successfully established for SIM1 and ZNF154. Correlation between a pathological cancer cell fraction and methylation levels obtained by pyrosequencing was confirmed to be high (R = 0.53 for SIM1, 0.64 for ZNF154). Finally, an independent validation cohort of 29 ovarian cancer FFPE samples was analyzed. ZNF154 methylation showed a high correlation with the pathological cancer cell fraction (R = 0.77, P < 0.0001). Therefore, the ZNF154 methylation level was considered to be useful for the estimation of ovarian cancer cell fraction, and is expected to help accurate molecular analysis.

Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids.

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs.

Stickler syndrome – lessons from a national cohort

In 2011 NHS England commissioned a new national specialist MDT service for patients and families affected by Stickler syndrome. The Stickler syndromes form part of the spectrum of inherited vitreoretinopathies and are the most common cause of retinal detachment in childhood and the most common cause of familial retinal detachment. Now in its 10th year, the Stickler Highly Specialised Service (HSS) has assessed 1673 patients from 785 families. Using a combination of accurate phenotyping and molecular genetic analysis it is possible to identify the underlying genetic mutation in over 95% of cases including those with deep intronic mutations likely to be missed by conventional exome panel analysis and which require whole gene sequencing and supplementary functional analysis to confirm pathogenicity. The vast majority that presents to ophthalmologists will be from one of three autosomal dominant sub-groups with a high associated risk of retinal detachment but the diagnosis is often overlooked, especially in adults. In contrast to many other blinding retinal conditions, blindness through giant retinal tear detachment particularly in children is largely preventable provided these high-risk groups are identified and appropriate evidence-based prophylaxis offered. This article summarises ten selected briefcase histories from the national dataset with key learning points from each.

Ultramorphology and Molecular Studies of Contracaecum Larvae (Nematoda: Anisakidae) Collected in Five Cyprinid Fish Species from Sulaimani Province, Kurdistan Region-Iraq.

SummaryA total of 1134 freshwater fishes belonging to Cyprinidae (Acanthobrama marmid (n=20), Alburnus caeruleus (n=7), Alburnus mossulensis (n=62), Arabibarbus grypus (n=123), Barbus lacerta (n=7), Capoeta trutta (n=222), C. umbla (n=161), Carasobarbus kosswigi (n=5), C. luteus (n=89), Carassius auratus (n=54), Chondrostoma regium (n=52), Cyprinion kais (n=10) and C. macrostomum (n=322)) were collected in different water bodies in Sulaimani Province, Kurdistan Region-Iraq for the presence of larval nematode of the genus Conteacaecum. This investigation revealed that 17 fishes belonged to five species (A. marmid, A. grypus, C. trutta, C. luteus and C. regium) were infected with Contracaecum larvae with prevalence of 35 %, 0.81 %, 0.90 %, 4.49 % and 5.76 %, respectively. The third- larval stage was morphologically studied by optical microscopy, and the ultrastructure was investigated using scanning electron microscopy (SEM). In addition, molecular analysis was carried out by amplifying, sequencing and comparing different gene loci, including internal transcribed spacers (ITS-1 and ITS-2) and cytochrome oxidase c subunit-II (COX-2), of the different isolated Contracaecum larvae. These sequences were also compared with closely related nematode sequences from the GenBank. Fifteen sequences were obtained for this study from the collected Contracaecum larvae. ITS-1, ITS-2 and COX-2 were amplified by polymerase chain reaction (PCR) and sequenced. The sequences of ITS-1, ITS-2 and COX-2 revealed that the collected Contracaecum larval specimens from all infected fish species represented one species (Contracaecum rudolphii B) based on the identity percentage in the GenBank database. The genetic characterisation of the parasite in the present study is available in the GenBank database, and the obtained ITS-1, ITS-2 and COX-2 sequences were deposited in GenBank. The present study provides information on the accurate identification and molecular analysis of Contracaecum larvae in the infected fish species in Sulaimani Province, Kurdistan Region-Iraq.

Enhanced Molecular Diagnosis of Bloodstream Candida Infection with Size-Based Inertial Sorting at Submicron Resolution.

Inertial microfluidics has been proven to be a powerful tool for high-throughput, size-based cell sorting in diverse biomedical applications. In the case of Candida-related sepsis, Candida species and major blood cells (i.e., red blood cells and white blood cells) have a size distribution of 3-5 and 6-30 μm, respectively. To effectively retrieve a majority of Candida species and remove most of the interfering blood cells for accurate molecular analysis, inertial sorting of micron-sized biological particles with submicron size difference is highly desired, but far unexplored till now. In this work, we present a new channel design for an inertial microfluidic sorting device by embedding microsquares to construct periodic contractions along a series of repeating curved units. This unique channel design allows us to enhance inertial lift force at the microsquare zone and produce localized secondary Dean flow drag force in addition to global Dean flow drag force. This inertial sorting device has successfully separated 5.5 μm particles from 6.0 μm particles with a recovery ratio higher than 80% and a purity higher than 92%, demonstrating a size-based inertial sorting at submicron resolution (i.e., 0.5 μm). We further applied this inertial sorting device to purify Candida species from whole blood sample for enhanced molecular diagnosis of bloodstream Candida infection and especially compared it with the commonly used lysis-centrifugation-based purification method (STEM method) by recovering two species of Candida (Cornus glabrata and Candida albicans) from Candida-spiked blood samples. Through quantitative polymerase chain reaction (qPCR) analysis, we found that our inertial sorting approach has nearly 3-fold improvement on the pathogen recovery than the STEM method at pathogen abundances of 103 cfu/mL and 102 cfu/mL. The present inertial sorting at submicron resolution provides a simple, rapid, and efficient pathogen purification method for significantly improved molecular diagnosis of bloodstream Candida infection.

Associations between IGF1, IGFBP2 and TGFß3 Genes Polymorphisms and Growth Performance of Broiler Chicken Lines.

Simple SummaryThe main goal of breeding programs for broiler chickens is to increase growth rate and breast and thigh muscles weight. The candidate gene approach is a powerful technique for genetically improving performance traits in chickens. We studied the associations of the single nucleotide polymorphisms of three genes involved in protein synthesis, glucose metabolism and cell proliferation (IGF1, IGFBP2, TGFβ3) with performance traits in the Hubbard F15 and Cobb E chicken lines. Based on our results, it can be concluded that the TGFβ3 gene could be used as a candidate gene marker for chicken growth traits.Marker-assisted selection based on fast and accurate molecular analysis of individual genes is considered an acceptable tool in the speed-up of the genetic improvement of production performance in chickens. The objective of this study was to detect the single nucleotide polymorphisms (SNPs) in the IGF1, IGFBP2 and TGFß3 genes, and to investigate their associations with growth performance (body weight (BW) and average daily gain (ADG) at 14, 21, 28, 35 and 42 days of age) and carcass traits in broilers. Performance (carcass) data (weight before slaughter; weights of the trunk, giblets, abdominal fat, breast muscle and thigh muscle; slaughter value and slaughter percentage), as well as blood samples for DNA extraction and SNP analysis, were obtained from 97 chickens belonging to two different lines (Hubbard F15 and Cobb E) equally divided between the two sexes. The genotypes were detected using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) methods with specific primers and restrictase for each gene. The statistical analysis discovered significant associations (p < 0.05) between the TGFβ3 SNP and the following parameters: BW at 21, 28 and 35 days, trunk weight and slaughter value. Association analysis of BWs (at 21, 28 and 35 days) and SNPs was always significant for codominant, dominant and overdominant genetic models, showing a possible path for genomic selection in these chicken lines. Slaughter value was significant for codominant, recessive and overdominant patterns, whereas other carcass traits were not influenced by SNPs. Based on the results of this study, we suggested that the TGFβ3 gene could be used as a candidate gene marker for chicken growth traits in the Hubbard F15 and Cobb E population selection programs, whereas for carcass traits further investigation is needed.

Complementary vibrational spectroscopy

Vibrational spectroscopy, comprised of infrared absorption and Raman scattering spectroscopy, is widely used for label-free optical sensing and imaging in various scientific and industrial fields. The two molecular spectroscopy methods are sensitive to different types of vibrations and provide complementary vibrational spectra, but obtaining complete vibrational information with a single spectroscopic device is challenging due to the large wavelength discrepancy between the two methods. Here, we demonstrate simultaneous infrared absorption and Raman scattering spectroscopy that allows us to measure the complete broadband vibrational spectra in the molecular fingerprint region with a single instrument based on an ultrashort pulsed laser. The system is based on dual-modal Fourier-transform spectroscopy enabled by efficient use of nonlinear optical effects. Our proof-of-concept experiment demonstrates rapid, broadband and high spectral resolution measurements of complementary spectra of organic liquids for precise and accurate molecular analysis.

The identification of new ATAD2 bromodomain inhibitors: the application of combined ligand and structure-based virtual screening

Ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches were used to identify new inhibitors for ATAD2 bromodomain. The LBVS approach was used to search 23,129,083 clean compounds to identify compounds similar to an active compound with reported pIC50 equal to 7.2. Based on LBVS results, 19 compounds were selected. To perform SBVS, by applying nine filters on 23,129,083 clean compounds, 1,057,060 compounds were selected. After performing SBVS on these selected compounds with idock software, 16 compounds with the lowest binding energies were selected. More accurate molecular docking analysis was performed on these 35 selected compounds by using iGEMDOCK software and six of them with the lowest binding energies were selected as hit compounds. These compounds were zinc36647229, zinc77969074, zinc13637358, zinc77971540, zinc12991296 and zinc19374204.

Leveraging Cheminformatics Strategies for Inorganic Discovery: Application to Redox Potential Design

Virtual high throughput screening, typically driven by first-principles, density functional theory calculations, has emerged as a powerful tool for the discovery of new materials. Although the computational materials science community has benefited from open source tools for the rapid structure generation, calculation, and analysis of crystalline inorganic materials, software and strategies to address the unique challenges of inorganic complex discovery have not been as widely available. We present a unified view of our recent developments in the open source molSimplify code for inorganic discovery. Building on our previous efforts in the automated generation of highly accurate inorganic molecular structures, first-principles simulation, and property analysis to accelerate high-throughput screening, we have recently incorporated a neural network that both improves structure generation and predicts electronic properties prior to first-principles calculation. We also provide an overview of how multimillion ...

FRET spectroscopy—towards effective biomolecular probing

The advent of super-resolution microscopy has been a major breakthrough in bioscience research, allowing accurate molecular signaling analysis of cellular and biological materials.

Abstract 5264: A novel strategy for accuurate and unambiguous mutation detection of the highly homologous PMS2 gene

Abstract Background: Heterozygous mutations in the DNA mismatch repair gene, PMS2 contribute to Lynch syndrome. Carrier individuals are at increased risk of developing cancers, including colorectal and endometrial cancers. Biallelic mutations in PMS2 can cause constitutional mismatch-repair-deficiency syndrome, which is characterized by childhood onset malignancy, including brain cancers, hematologic malignancies, colorectal cancers, and multiple intestinal polyposis. Molecular analysis of the PMS2 gene is complicated by the presence of a large family of highly homologous sequences in other genomic regions and frequent gene conversion between the active gene and pseudogenes. Although the combination of multiplex ligation-dependent probe amplification (MLPA) assay and Sanger sequencing can largely overcome this problem, copy number variations (CNVs) at the 3′ end cannot be unambiguously differentiated when gene conversion is involved. Here we described a novel approach that combines long range PCR (LR-PCR), next generation sequencing (NGS), and MLPA to identify sequence and copy number changes of the PMS2 gene. Method: A comprehensive workflow incorporating MLPA, LR-PCR, and NGS was carefully designed for sequence and CNV analyses of the PMS2 gene. MLPA was used to detect CNVs in PMS2 and PMS2CL. In order to distinguish mutations in the active gene from changes in the pseudogene, the active gene was specifically enriched by LR-PCR, followed by whole gene NGS analysis to detect point mutations and CNVs. CNVs can be further confirmed by MLPA. Allele dropout is overcome by capture-based NGS. Results: Using the comprehensive workflow described above, we have identified CNVs in all coding exons of the PMS2 gene: exonic deletions (E2-E9, E5-E9, E8, E10, E14), exonic duplications (E11-E12), whole gene deletion (E1-E15), and a nonsense mutation, S22X. We identified an exon 14 deletion (c.2276-113_c.2445+1596del) in the active gene that could not be differentiated originally by the combination of MLPA and Sanger sequencing due to a gene conversion event in the 3′ region between PMS2 and PMS2CL, however, with LR-PCR followed by NGS, the deletion was unequivocally identified. Breakpoint analysis of multiple samples with exon 14 deletions revealed that frequent exon 14 deletions were mediated by homologous Alu sequences. We also identified a potential LINE1 sequence mediated intragenic duplication flanking exons 11 and 12. Conclusion: The combined strategies of long range PCR, MLPA, and deep coverage NGS ensure accurate and unambiguous identification of a wide spectrum of mutations, including point mutations, small indels, and copy number changes, in addition to the determination of breakpoints. The novel strategy of comprehensive workflow provides a reliable tool for NGS based accurate molecular analysis of genes containing multiple copies of highly homologous sequences such as PMS2. Citation Format: Jianli Li, Hongzheng Dai, Yangming Feng, Jia Tang, Stella Chen, Xia Tian, Elizabeth Gorman, Eric S. Schmitt, Jing Wang, Victor Wei Zhang, Lee-Jun Wong. A novel strategy for accuurate and unambiguous mutation detection of the highly homologous PMS2 gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2015-5264

THz extended spectrum of the monodeuterated methyl formate (DCOOCH3)

Context. Laboratory spectral recordings and an accurate molecular spectral analysis of any potential interstellar molecule are essential for generating a complete spectroscopic line list. This permits predicting the frequencies and intensities of any transition so that subsequently, it can be identified in the interstellar medium. Aims. Our analysis of DCOOCH3 aims to provide a comprehensive spectral catalog that encompasses as much as possible the frequency coverage of the new-generation far-IR and submillimeter wave observation facilities. Methods. We newly measured the rotational spectrum of DCOOCH3 in the laboratory of the Jet Propulsion Laboratory in the frequency range of 0.85 to 1.5 THz. We jointly analyzed the new data with literature data using the rho axis method, which is a tool developed for the spectral analysis of molecules with large-amplitude internal CH3 rotors. Results. We fit 27 spectroscopic constants of DCOOCH3 to 3763 transitions with highest values of J = 69 and Ka = 36 of the ground torsional state with a standard (unitless) deviation of 0.97. With respect to previous work, this is a significantly better result that was obtained with 2060 more transitions, and we also achieved a better accuracy for the new parameter values.

Intratumor heterogeneity of DNA copy number aberrations in gastric and oesophageal adenocarcinomas.

78 Background: DNA copy number aberrations (CNAs) are common in oesophageal and gastric adenocarcinomas (OGCs) and display extensive inter-tumour heterogeneity. CNA patterns define gastric cancer molecular subtypes and ERBB2 amplifications, present in a small fraction of patients with OGC, are predictive for ERBB2-targeted drug sensitivity. Together, this suggests a critical role of CNAs determining OGC tumour biology and clinical outcomes. Despite this, predictive and prognostic CNA biomarkers have not been identified for the majority of OGCs, precluding the development of effective personalized therapy approaches for these aggressive tumours. Intra-tumour heterogeneity, characterized by the presence of multiple subclones with distinct genetic profiles within an individual cancer, can hinder the accurate molecular analysis and classification of tumours. The aim of this pilot study was to assess whether chemotherapy-naïve localized OGCs display intra-tumour macroheterogeneity of CNA profiles. Methods: Tissue specimens from four tumour regions representing the macroscopic spatial extent of each of five OGCs were systematically collected after surgical resection. DNA extracted from these FFPE specimens was analysed by molecular inversion probe SNP arrays for high resolution CNA detection. Results: Comparison of genome wide copy number and B-allele frequency profiles suggested highly concordant CNA profiles across the regions from individual primary tumours. Eight driver CNAs leading to amplification of the MET, KRAS, ERBB2, PIK3CA or FGFR2 oncogenes were identified in 4/5 tumours. Only one out of these eight driver CNA’s, harbouring the KRAS oncogene, was heterogeneous within a tumour. Conclusions: Although chromosomal instability is thought to be common in this tumour type, this pilot study suggests that macroheterogeneity is limited and that CNA profiles assessed from a single tumour biopsy are likely to be representative of the dominant CNA profile of localized OGCs. Thus, clinical correlative CNA analyses may be possible from single biopsies of localized OGCs. Mutational heterogeneity and microheterogeneity in microdissected and single cells are currently being investigated.

Assessing minimal tumour cell requirements and DNA input for accurate molecular analysis of FFPE tissues using MALDI-TOF MS

Aim Determine in FFPE core biopsies and cell blocks, the minimal tumour cell count and DNA input to accurately detect mutations using MALDI- TOF mass spectrometry (MS) platform and Sequenom panel profiling >200 mutations in EGFR, BRAF, KRAS, NRAS and cKIT. Methods Core biopsies and cell blocks were quantitated for tumour cells/hpf in cases with and without mutations. Mean tumour cell number/5hpf was quantified representing 20% of total cell block/core biopsy volume. Student t-test compared tumour cell numbers from samples with and without mutations. Fractional unextended primer (UEP) versus input DNA was recorded against dilutions of DNA. Results Mean tumour cell count was 98 (range 10–335) and 126/hpf (range 28–214) respectively and was not significantly different when compared with mutation negative cases. Dilution data showed that 0.91 to 1.00 of UEP used in all reactions containing DNA template (range 4–40 ng). 0.01 UEP used in assays with no DNA template. Allelic ratio 5%. Discussion MALDI-TOF MS platform accurately detects mutations to a level of 98 tumour cells/hpf (mean) of FFPE sections and is accurate up to a total input of DNA to 4 ng. This cut off is important for accurate diagnosis of molecular mutations in specimens with minimal tumour cells.

Tools to assess tissue quality

Biospecimen science has recognized the importance of tissue quality for accurate molecular and biomarker analysis and efforts are made to standardize tissue procurement, processing and storage conditions of tissue samples. At the same time the field has emphasized the lack of standardization of processes between different laboratories, the variability inherent in the analytical phase and the lack of control over the pre-analytical phase of tissue processing. The problem extends back into tissue samples in biorepositories, which are often decades old and where documentation about tissue processing might not be available. This review highlights pre-analytical variations in tissue handling, processing, fixation and storage and emphasizes the effects of these variables on nucleic acids and proteins in harvested tissue. Finally current tools for quality control regarding molecular or biomarker analysis are summarized and discussed.

Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch (®) technology.

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology.

Functional analysis of 11 novel GBA alleles

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or 'de novo' mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.