Paraffin-embedded Clinical Samples Research Articles

Single Nucleus Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Gliomas.

Gliomas, the predominant form of brain cancer, comprise diverse malignant subtypes with limited curative therapies available. The insufficient understanding of their molecular diversity and evolutionary processes hinders the advancement of new treatments. Technical complexities associated with formalin-fixed paraffin-embedded (FFPE) clinical samples hinder molecular-level analyses of gliomas. Current single-cell RNA sequencing (scRNA-seq) platforms are inadequate for large-scale clinical applications. In this study, automated snRandom-seq is developed, a high-throughput single-nucleus total RNA sequencing platform optimized for archival FFPE samples. This platform integrates automated single-nucleus isolation and droplet barcoding systems with the random primer-based scRNA-seq chemistry, accommodating a broad spectrum of sample types. The automated snRandom-seq is applied to analyze 116492 single nuclei from 17 FFPE samples of various glioma subtypes, including rare clinical samples and matched primary-recurrent glioblastomas (GBMs). The study provides comprehensive insights into the molecular characteristics of gliomas at the single-cell level. Abundant non-coding RNAs (ncRNAs) with distinct expression profiles across different glioma clusters and uncovered promising recurrence-related targets and pathways in primary-recurrent GBMs are identified. These findings establish automated snRandom-seq as a robust tool for scRNA-seq of FFPE samples, enabling exploration of molecular diversities and tumor evolution. This platform holds significant implications for large-scale integrative and retrospective clinical research.

High-resolution and quantitative spatial analysis reveal intra-ductal phenotypic and functional diversification in pancreatic cancer.

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRASG12D . The basal-like mRNA panel better captured the basal-like cell states than widely used protein markers. The panels corroborated the co-existence of the classical and basal-like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial-to-mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin-positive cells in the tumour. Uneven spatial distribution of cancer-associated fibroblasts could recreate similar intra-organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Improved NGS-based detection of microsatellite instability using tumor-only data.

Microsatelliteinstability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

High-Throughput NanoString Analysis of Oncogenic Human Papillomavirus and Tumor Microenvironment Transcription in Head and Neck Squamous Cell Carcinoma.

Human papillomaviruses (HPVs), specifically high-risk HPVs, are responsible for up to 3% of all cancers in women and up to 2% of all cancers in men. They have been identified as the etiological agent of cervical cancer and have been increasingly found to be the driver behind head and neck cancers of the oropharynx. A system in which we can simultaneously observe transcriptional changes to both a host's tumor microenvironment and its associated oncogenic driver (e.g., HPV) would be highly valuable for understanding HPV's role in tumorigenesis. This article describes a detailed methodology for utilizing high-throughput RNA analysis to study viral transcription in formalin-fixed, paraffin-embedded clinical tumor samples. Although our lab utilizes these methods for the study of head and neck cancer, the principles contained within are widely applicable to all fields of HPV study. © 2021 Wiley Periodicals LLC. Basic Protocol: HPV16 transcript analysis using NanoString Support Protocol 1: Preparation of RNA from formalin-fixed, paraffin-embedded slides Support Protocol 2: Preparation of RNA from cell lysates Support Protocol 3: Fluorometric RNA concentration and RNA integrity analysis Support Protocol 4: Determination of input RNA based on DV300 calculation.

Prognostic value of a hypoxia-related microRNA signature in patients with colorectal cancer.

Hypoxia has been particularly associated with poor prognosis in cancer patients. Recent studies have suggested that hypoxia-related miRNAs play a critical role in various cancers, including colorectal cancer (CRC). In the present study, we found 52 differentially expressed miRNAs in HT-29 cells under hypoxic conditions versus normoxic conditions by analyzing the profiles of miRNAs. Using Cox model, we developed a hypoxia-related miRNA signature consisting of four miRNAs, which could successfully discriminate high-risk patients in the Cancer Genome Atlas (TCGA) training cohort (n=381). The prognostic value of this signature was further confirmed in the TCGA testing cohort (n=190) and an independent validation cohort composed of formalin-fixed paraffin-embedded clinical CRC samples (n=220), respectively. Multivariable Cox regression and stratified survival analysis revealed this signature was an independent prognostic factor for CRC patients. Time-dependent receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of this signature was significantly larger than that of any other clinical risk factors or single miRNA alone. A nomogram was constructed for clinical use, which incorporated both the miRNA signature and clinical risk factors and performed well in the calibration plots. Collectively, this novel hypoxia-related miRNA signature was an independent prognostic factor, and it possessed a stronger predictive power in identifying high-risk CRC patients than currently used clinicopathological features.

Genome-wide identification of a novel miRNA-based signature to predict recurrence in patients with gastric cancer.

The current tumor node metastasis (TNM) staging system is inadequate for identifying high‐risk gastric cancer (GC) patients. Using a systematic and comprehensive‐biomarker discovery and validation approach, we attempted to build a microRNA (miRNA)‐recurrence classifier (MRC) to improve the prognostic prediction of GC. We identified 312 differentially expressed miRNAs in 446 GC tissues compared to 45 normal controls by analyzing high‐throughput data from The Cancer Genome Atlas (TCGA). Using a Cox regression model, we developed an 11‐miRNA signature that could successfully discriminate high‐risk patients in the training set (n = 372; P < 0.0001). Quantitative real‐time polymerase chain reaction‐based validation in an independent clinical cohort (n = 88) of formalin‐fixed paraffin‐embedded clinical GC samples showed that MRC‐derived high‐risk patients succumb to significantly poor recurrence‐free survival in GC patients (P < 0.0001). Cox and stratification analysis indicated that the prognostic value of this signature was independent of clinicopathological risk factors. Time‐dependent receiver operating characteristic (ROC) analysis revealed that the area under the curve of this signature was significantly larger than that of TNM stage in the TCGA (0.733 vs. 0.589 at 3 years, P = 0.004; 0.802 vs. 0.635 at 5 years, P = 0.005) and validation cohort (0.835 vs. 0.689 at 3 years, P = 0.003). A nomogram was constructed for clinical use, which integrated both MRC and clinical‐related variables (depth of invasion, lymph node status and distance metastasis) and did well in the calibration plots. In conclusion, this novel miRNA‐based signature is superior to currently used clinicopathological features for identifying high‐risk GC patients. It can be readily translated into clinical practice with formalin‐fixed paraffin‐embedded specimens for specific decision‐making applications.

Assessment of alternatives to environmental toxic formalin for DNA conservation in biological specimens.

One essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin-fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservation using an artificial ageing treatment and followed the evolution of specimens' DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

N-Glycan matrix-assisted laser desorption/ionization mass spectrometry imaging protocol for formalin-fixed paraffin-embedded tissues.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of the proteome of a tissue has been an established technique for the past decade. In the last few years, MALDI-MSI of the N-glycome has emerged as a novel MALDI-MSI technique. To assess the accuracy and clinical significance of the N-linked glycan spatial distribution, we have developed a method that utilises MALDI-MSI followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) in order to assign glycan structures to the differentiating MALDI-MSI glycan masses released from the tissue glycoproteins. Our workflow presents a comprehensive list of instructions on how to (i) apply MALDI-MSI to spatially map the N-glycome across formalin-fixed paraffin-embedded (FFPE) clinical samples, (ii) structurally characterise N-glycans extracted from consecutive FFPE tissue sections by LC/MS/MS, and (iii) match relevant N-glycan masses from MALDI-MSI with confirmed N-glycan structures determined by LC/MS/MS. Our protocol provides groups that are new to this technique with instructions how to establish N-glycan MALDI-MSI in their laboratory. Furthermore, the method assigns N-glycan structural detail to the masses obtained in the MALDI-MS image. Copyright © 2017 John Wiley & Sons, Ltd.

Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples.

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intra-tumor genetic heterogeneity poses a significant challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Given the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh/frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ display intra-tumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

1174P - Detection of NRAS, KRAS and BRAF mutations in FFPE derived DNA with a novel targeted resequencing-based diagnostics assay

Identification of cancer-associated mutations has become standard care for cancer treatment. Somatic mutations in NRAS, KRAS and BRAF genes are important to decide the treatment options for patients with cancer. Mutations in NRAS, KRAS or BRAF help to determine the treatment options for patients when EGFR inhibitor or BRAF inhibitor treatment is considered. The novel SOMATIC 1 MASTRTM Plus Dx® assay has been developed to allow analysis of the hotspot mutations or of the full coding region of KRAS, NRAS and BRAF genes in combination with Next Generation Sequencing (NGS). To assess the performance characteristics of the SOMATIC 1 MASTRTM Plus Dx® assay, a multicenter study was performed. The performance of SOMATIC 1 MASTR Plus Dx to detect single nucleotide variants (SNV) and small insertions and/or deletions (indels) in the NRAS, KRAS and BRAF genes at a variant allele frequency (VAF) as 5% in the entire coding region and for specific hotspot mutations was evaluated. The sample population comprised 252 formalin-fixed, paraffin-embedded (FFPE) clinical samples derived from a variety of different cancer types and 4 proficiency samples (Tru-Q Reference Standards, Horizon Discovery). Amplicon libraries were analysed using Illumina MiSeq platform. Data analysis was performed with the SeqNext module (JSI Medical Systems) and the Sophia DDM platform (ILL1MR1S5_somatic1_v2). SOMATIC 1 MASTRTM Plus Dx® showed amplification uniformity of 97.8% (% of amplicons covered at 0.2x of the mean coverage) and a target specificity of 99.1%. The overall limit of detection (LOD) of the assay was determined as 2% VAF, and showed to be as low as 1% for hotspot mutations. Diagnostically, the assay showed 99.96% (95 % CI ≥ 99.90 %) accuracy, 99.96% (95 % CI ≥ 99.90 %) specificity and 100% (95 % CI ≥ 98.54 %) sensitivity. Furthermore, Somatic 1 MASTRTM Plus Dx® assay showed to be 99.99% (95 % CI ≥ 99.97 %) repeatable and 99.96% (95 % CI ≥ 99.91%) reproducible. We have established a targeted and cost-effective NGS assay for the detection of KRAS, NRAS, and BRAF somatic mutations in clinical FFPE samples that can be routinely incorporated into clinical practice.

Recent advances in mass spectrometry analysis of histone post-translational modifications: potential clinical impact of the PAT-H-MS approach

SUMMARYHistone post-translational modifications (hPTMs) contribute to the regulation of gene expression and increasing evidence links them to the development of various pathologies, highlighting their potential as biomarkers for prognostic, diagnostic and therapeutic applications. Mass spectrometry (MS) has emerged as a powerful analytical tool for hPTM analysis, which has also been applied to the analysis of epigenetic aberrations in diseases. However, the potential offered by the MS-based hPTM analysis of clinical samples for epigenetic biomarker discovery has been left largely unexploited. This article summarizes the contribution of MS-based approaches to clinical epigenetics, with a special focus on the PAThology tissue analysis of Histones by Mass Spectrometry (PAT-H-MS) approach – which represents the first application of MS-based hPTM analysis to formalin-fixed paraffin-embedded clinical samples – discussing its strengths and limitations, as well as possible implementations.

MicroRNA-21 expression and its pathogenetic significance in cutaneous melanoma.

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expression but not with cytoplasmic PTEN expression. An anti-miR-21 preferentially altered nuclear PTEN in melanoma cell lines. The presence of a BRAF or NRAS mutation had no significant effect on miR-21 expression. These data suggest miR-21 may exert an oncogenic effect in melanoma by favouring redistribution of PTEN to the nucleus.

Abstract 4886: Repair of challenging FFPE DNA improves library success rate and sequencing quality

Abstract Formalin-fixed, paraffin-embedded (FFPE) clinical samples are an invaluable source of information about genetic alterations in human disease, especially cancer. Next generation sequencing is a powerful tool to mine that information. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. In this study, we investigated the effects of DNA repair on library preparation and sequencing from FFPE samples. We evaluated a repair enzyme mix that is designed to work on a broad range of DNA damage including modified bases, nicks, gaps, and blocked 3′ ends. Results from FFPE DNA samples of varying quality show that DNA repair generally increases library yields by 10-170%, with the largest improvement on samples of poor quality. Careful analysis of sequencing data shows that base calling qualities for all 4 bases are improved upon DNA repair. Aberrant G:C to A:T mutation were significantly reduced, consistent with cytosine deamination being induced during fixation and storage at room temperature. Interestingly, sequencing miscalls for base pair changes not typically associated with fixation were also reduced. We are currently investigating the source of these mutations. With DNA repair a noticeably positive impact on read mapping and read pairing was observed, resulting in the generation of more useable data. Pretreatment of FFPE DNA by the repair enzyme mix is easily implemented upstream of library construction, allowing fast turnaround time and easy automation. We expect that these improvements will enable the analysis of many FFPE samples that would otherwise not be accessible. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis, Thomas Evans. Repair of challenging FFPE DNA improves library success rate and sequencing quality. [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 4886. doi:10.1158/1538-7445.AM2015-4886

Prostate tumor overexpressed 1 is a novel prognostic marker for hepatocellular carcinoma progression and overall patient survival.

The gene prostate tumor overexpressed 1 (PTOV1) was first found to be upregulated in prostate cancer. This upregulation increased tumor cell proliferation, retinoic acid resistance, and migration. This study investigated the expression and prognostic significance of PTOV1 in hepatocellular carcinoma (HCC).Real-time Polymerase Chain Reaction and western blot analysis were performed to examine PTOV1 expression in 11 HCC cell lines and 2 normal hepatic cell lines. PTOV1 expression levels were also determined in 8 pairs of tissue samples taken from primary HCC tumors and the matched adjacent noncancerous liver tissue from the same patient. Immunohistochemistry assays assessed PTOV1 protein expression in paraffin-embedded clinical samples taken from 215 HCC patients. The correlation of PTOV1 expression with the clinicopathological parameters was evaluated along with the prognostic impact of PTOV1 expression in these HCC patients.PTOV1 mRNA and protein were overexpressed in HCC cell lines compared with normal liver cell lines and were overexpressed in primary HCC samples compared with the matched noncancerous liver tissue samples. In the paraffin-embedded tissue samples from 215 HCC patients, PTOV1 protein expression was significantly correlated with T classification, N classification, clinical stage, and serum α-fetoprotein. HCC patients with higher PTOV1 expression had shorter survival times than patients with lower PTOV1 expression.Our study demonstrated that PTOV1 overexpression is correlated with increased aggressiveness of HCC and could be a prognostic biomarker for patients with HCC.

Abstract 5199: Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer

Abstract Recently, genome organizer protein special AT-rich sequence-binding protein 1(SATB1) has been reported to promote a metastatic phenotype and correlate with poor prognosis in breast cancer. The mechanism of SATB1 elevation in breast cancer remains elusive. This study was to investigate whether microRNAs (miRNAs) play a role in modulating SATB1 expression in ductual breast cancer by using archival formalin-fixed and paraffin-embedded (FFPE) clinical samples. Firstly, four canonical target prediction databases, including TargetScan, PicTar, miRanda and miRDB, were utilized to predict the interaction between miRNA and the 3′-UTR of SATB1 mRNA. The results of the prediction software programs were then integrated by displaying Venn diagram. Secondly, the Qiagen miRNeasy FFPE kit was used to isolate total RNA from 93 specimens with primary invasive ductual breast cancer. Expression of candidate miRNA transcriptional levels was measured using TaqMan MicroRNA Assays with miRNA-specific stem loop primers by relative real-time quantitative RT-PCR. The relative levels of miRNA expression were calculated from the relevant signals by normalization with the signal for RNU6B miRNA expression in each sample. Meanwhile, SATB1 protein expression levels were assessed by immunohistochemistry(IHC). Finally, correlations between the expression levels of these miRNAs and SATB1 were analyzed. Overall 22 miRNAs, 24 miRNAs, 76 miRNAs and 75 miRNAs were found by TargetScan, PicTar, miRanda and miRDB, to target SATB1 respectively, whereas only miR-7, miR-21, miR-23a, miR-23b, miR-34a and miR-155 were found by all four programs to be predicted to regulate SATB1. Quantitative RT-PCR detection analysis showed that expression levels of miR-34a were much higher in SATB1-negative than in SATB1-positive tumors (p&amp;lt;0.001). whereas lower miR-7 expression showed a strong trend toward an association with elevated SATB1 expression (P = 0.056). We then extended the analysis of miR-34a expression in breast cancer tissues to include all scores for SATB1 protein expression, assessed semiquantitatively by IHC. miR-34a expression levels were gradually decreased as SATB1 protein scores increased. Moreove, our quantitative RT-PCR results showed no differences in miR-21, miR-23a, miR-23b, and miR-155 expression between SATB1-negative and SATB1-positive tumors. We thus conclude that SATB1 maybe a novel target of miR-34a in human breast cancer. Downregulation of miR-34a promotes human breast cancer to progress through reduced repression of SATB1. Restoration of miR-34a may prove therapeutic in breast cancer patients in which aberrant SATB1 expression plays a role. Note: This abstract was not presented at the meeting. Citation Format: Weiming Duan, Zixing Chen, Li Yao, Min Tao, Kai Chen, Wei Liu, Jiannong Cen. Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2014-5199

High-Throughput Detection of Clinically Relevant Mutations in Archived Tumor Samples by Multiplexed PCR and Next-Generation Sequencing

Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples. We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer. We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.

Utility of the QIAGEN/ Artus PCR Assays and Meningofinder™ for the Detection of Human Herpes Virus Genomes in Formalin-Fixed Paraffin-Embedded Clinical Samples

ABSTRACT: Aim: The aim of the study was to analyze whether human herpes viruses can be detected from formalin-fixed paraffin-embedded (FFPE) tissues with two commercial methods. Materials &amp; methods: In total, 91 clinical FFPE samples were analyzed with QIAGEN/artus (Hilden, Germany) qPCRs and the Meningofinder™ assay from PathoFinder (Maastricht, The Netherlands). Results: A total of 27 out of 91 samples were tested for HSV-1 and HSV-2. Three of these 27 samples (11.1%) were positive for HSV-1 and one sample was positive for HSV-2. Out of 91 samples, 50 were tested for CMV, of which six samples were positive (12%). In total, 12 out of 91 samples were tested for the presence of Varizella zoster virus DNA, revealing one as positive (8%). Finally, two samples were tested negative for EBV. Conclusion: Minor differences were identified between both assays, making them a reliable tool for detection of herpes viruses from FFPE material, although this is an off-label use for both assays.

Shifted termination assay (STA) fragment analysis to detect BRAF V600 mutations in papillary thyroid carcinomas

BackgroundBRAF mutation is an important diagnostic and prognostic marker in patients with papillary thyroid carcinoma (PTC). To be applicable in clinical laboratories with limited equipment, diverse testing methods are required to detect BRAF mutation.MethodsA shifted termination assay (STA) fragment analysis was used to detect common V600 BRAF mutations in 159 PTCs with DNAs extracted from formalin-fixed paraffin-embedded tumor tissue. The results of STA fragment analysis were compared to those of direct sequencing. Serial dilutions of BRAF mutant cell line (SNU-790) were used to calculate limit of detection (LOD).ResultsBRAF mutations were detected in 119 (74.8%) PTCs by STA fragment analysis. In direct sequencing, BRAF mutations were observed in 118 (74.2%) cases. The results of STA fragment analysis had high correlation with those of direct sequencing (p < 0.00001, κ = 0.98). The LOD of STA fragment analysis and direct sequencing was 6% and 12.5%, respectively. In PTCs with pT3/T4 stages, BRAF mutation was observed in 83.8% of cases. In pT1/T2 carcinomas, BRAF mutation was detected in 65.9% and this difference was statistically significant (p = 0.007). Moreover, BRAF mutation was more frequent in PTCs with extrathyroidal invasion than tumors without extrathyroidal invasion (84.7% versus 62.2%, p = 0.001). To prepare and run the reactions, direct sequencing required 450 minutes while STA fragment analysis needed 290 minutes.ConclusionsSTA fragment analysis is a simple and sensitive method to detect BRAF V600 mutations in formalin-fixed paraffin-embedded clinical samples.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5684057089135749