Normal Formalin-fixed Paraffin-embedded Research Articles

Single-molecule localisation microscopy (SMLM) is feasible in human and animal formalin fixed paraffin embedded (FFPE) tissues in medical renal disease

AimsEstablishment of a protocol for routine single-molecule localisation microscopy (SMLM) imaging on formalin fixed paraffin embedded (FFPE) tissue using medical renal disease including minimal change disease (MCD) and focal segmental...

Abstract 5021: Circulating mutated and methylated tumor DNA detection for colorectal cancer diagnosis

Abstract Introduction: Detection of mutation and methylation patterns in liquid biopsies has emerged as a promising approach for screening early-stage cancers. We have developed a targeted next-generation sequencing (NGS)-based assay “OptiSeqTM Colorectal Cancer Detection Panel”, a combined platform for assessing mutation and methylation in both low-quantity (plasma cfDNA) and low-quality formalin-fixed paraffin-embedded (FFPE) tissue for colorectal cancer (CRC) detection. Methods: The OptiSeqTM Colorectal Cancer Detection Panel targets 442 SNVs and 434 CpG sites to detect circulating tumor DNA (ctDNA) in early-stage patients. With this panel, we profiled 6 early-stage and 6 metastatic CRC samples (matching cfDNA and FFPE) along with 2 primary colon specimens with adjacent normal tissue DNA. We also profiled 2 matching cfDNA and FFPE samples from precancerous adenomas (advanced adenomas (AA)) and 6 AA FFPE tissue DNA. In addition, we characterized the mutation and methylation pattern in 10 CRC fresh-frozen tissue DNA samples. For healthy individuals, we profiled 12 normal colon FFPE samples and 10 plasma cfDNA. The clinical performance for the CRC detection panel was calculated using AA, CRC, and cancer-free individuals. Results: For CRC early and metastatic matching cfDNA and FFPE samples, clinical samples testing showed that the CRC detection panel had a sensitivity of 100% (95% CI: 69.8-100.0) and specificity of 100% (95% CI: 81.5-100). For precancerous AA FFPE tissues, combined DNA mutation and methylation increased the CRC detection sensitivity to 100% (95% CI: 51.6-100.0) and specificity to 100% (95% CI: 61.8-100). More clinical samples testing is underway. Conclusion: We have developed the targeted NGS-based panel to detect clinically relevant mutation and methylation patterns and demonstrate its potential using the combined genetic and epigenetic markers making it a highly sensitive assay for early CRC diagnosis. Citation Format: Pushpinder Kaur Bains, Mikhail Kulak, Zhen Cui, Alisha Babu, Stella Tran, Aiguo Zhang, Michael Sha. Circulating mutated and methylated tumor DNA detection for colorectal cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5021.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

Chromogenic in situ hybridization reveals specific expression pattern of long non-coding RNA DRAIC in formalin-fixed paraffin-embedded specimen

Long non-coding RNA (lncRNA) plays an important role in the regulation of gene expression in normal and cancer cells. We previously discovered a novel tumor-suppressive lncRNA, DRAIC, in prostate cancer cells. Subsequent studies have demonstrated that DRAIC is dysregulated in various malignancies and exhibits a tumor-suppressive or pro-oncogenic function. However, details regarding its expression pattern in normal and cancerous tissues remain largely unknown. In this study, we performed chromogenic in situ hybridization (CISH) using RNAscope technology to assess DRAIC expression in formalin-fixed paraffin-embedded (FFPE) specimens. In the neuroendocrine-differentiated cancer cell line VMRC-LCD, CISH revealed a diffuse localization of DRAIC in the cytoplasm as well as specific accumulation in the nuclear compartment. DRAIC expression was comprehensively analyzed using tissue microarrays containing 89 normal and 155 tumor tissue samples. DRAIC was weakly expressed in normal epithelial cells of the colon, bronchiole, kidney, prostate, and testis. Conversely, DRAIC was moderately to highly expressed in some cancer tissues, including prostate adenocarcinoma, invasive ductal carcinoma of the breast, neuroendocrine carcinoma of the esophagus, lung adenocarcinoma, and small cell lung carcinoma. While DRAIC knockdown did not affect VMRC-LCD cellular viability and invasive ability, gene expression related to the neuroendocrine and cancer-related pathways was altered. Our expression analysis revealed the specific expression pattern of DRAIC in normal and cancerous FFPE tissues. The results presented here may lead to the elucidation of additional novel functions of DRAIC.

Abstract 2302: Detection of tumor-associated methylation and mutation signatures for early colorectal cancer diagnosis

Abstract Introduction: The ability to identify epigenetic signatures, together with genetic alterations, has expanded the translational utility of genomic sequencing in clinical settings. Epigenetic changes may appear at very early stages before diagnosis. Thus, they could be used as epigenetic biomarkers for early detection or risk of cancer development. Circulating cell-free DNA (cfDNA) profiling has emerged as a promising biomarker for cancer diagnosis and prognosis. Herein, we developed targeted next-generation sequencing (NGS) based assays for assessing multiple DNA methylated and mutated regions in both low-quantity (plasma cfDNA) and low-quality formalin-fixed paraffin-embedded (FFPE) tissue for colorectal cancer (CRC) detection. Methods: For the OptiSeqTM CRC methylation panel, we assessed the methylation status of CpG dinucleotides in 42 genes. With this panel, we profiled 6 CRC samples (matching cfDNA and FFPE), 2 primary colon tumors with adjacent normal, and 6 advanced adenomas (AA) FFPE tissue DNA. For healthy individuals, we profiled 12 normal colon FFPE samples and 31 plasma cfDNA (26 matching buffy coat DNA samples). For the OptiSeqTM CRC mutation panel, the mutation status was assessed in 43 genes. Here, we profiled 6 CRC samples (matching cfDNA and FFPE), 19 CRC plasma cfDNA, 4 AA FFPE and 42 healthy individuals’ plasma cfDNA (26 matching buffy coat DNA samples). The analytical and clinical performance for both panels (separately and combined) were calculated using advanced adenoma (AA), CRC, and cancer-free individuals. Results: For CRC cfDNA samples, clinical samples testing showed that the methylation panel had a sensitivity of 50% (95% CI: 13.9-86.0) and specificity of 100% (95% CI: 86.2-100) while the mutation panel yielded the sensitivity 88% (95% CI: 67.7-96.8) and specificity 100% (95% CI: 89.5-100). Combining the mutation panel with the methylation panel, the sensitivity increased to 100% (95% CI: 51.6-100) and specificity to 100% (95% CI: 65.1-93.3). For AA FFPE samples, the methylation panel analysis yielded a sensitivity of 100% (95% CI: 39.5-100.0) and a specificity of 100% (95% CI: 67.8-100). Conclusion: We have developed and validated the targeted NGS-based panels to detect clinically relevant mutation and methylation patterns and demonstrate its potential as a highly sensitive assay for early CRC diagnosis. Citation Format: Pushpinder Bains, Mikhail Kulak, Zhen Cui, Alisha Babu, Stella Tran, Bongyong Lee, Aiguo Zhang, Michael Sha. Detection of tumor-associated methylation and mutation signatures for early colorectal cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2302.

BAT25, ACVR2, and TGFBR2 Mononucleotide STR Markers: A Triplex Panel for Microsatellite Instability Testing in Colorectal Tumors.

Microsatellite instability (MSI) in colorectal cancer (CRC) patients is considered as a diagnostic and prognostic marker. MSI is a consequence of mismatch repair deficiency which is evaluated using the different microsatellite markers on the whole genome. In this pilot study, the diagnostic value of a novel triplex panel including three mononucleotide markers has been evaluated in comparison to the standard Promega kit for MSI testing in CRC patients with Amsterdam II criteria. DNA extracted from tumors and normal Formalin-Fixed Paraffin-Embedded (FFPE) tissues of index cases from 37 HNPCC (Hereditary non-polyposis colorectal cancer) families were evaluated for MSI state. Primer design for three markers, including BAT25, ACVR2, and TGFBR2, was performed using 19 nucleotides of the M-13 phage. The instability of each marker was assessed through fragment analysis in comparison with Promega kit markers for all patients. The sensitivity and specificity of each marker have been calculated. The comparative evaluation of MSI in both tumors and normal adjacent FFPE tissues demonstrated a separate sensitivity as 100%, 83.3%, and 76.9% for BAT25, ACVR2, and TGFBR2, respectively, and 100% sensitivity in the form of a triplex. Moreover, the specificity for each of these three markers in MSI testing was estimated as 100%, separately and in the form of the triplex in comparison with the Promega pentaplex standard Kit. A high sensitivity and specificity for the novel triplex panel in MSI-testing were estimated among Iranian patients. More studies are recommended to confirm this panel as a diagnostic kit for MSI testing.

Abstract 2347: Metabolomics profiling of formalin-fixed paraffin-embedded prostate tissues

Abstract The metabolic landscape of the prostate changes with malignant transformation, and metabolomics is a means of identifying diagnostic and prognostic biomarkers for prostate cancer. Epidemiology and registry studies containing archival formalin-fixed paraffin-embedded (FFPE) specimens are a rich resource for this work but this method of tissue preservation presents a challenge due to loss of metabolites. We optimized global metabolic profiling in FFPE tissue and identified metabolites distinguishing tumor from normal prostate tissue, and aggressive from non-aggressive prostate cancer. For optimization of metabolomics in FFPE tissues, we obtained matched frozen and FFPE tumor tissue from 62 prostate cancer cases who underwent radical prostatectomy (RP) at the University of North Carolina (UNC) hospital, of which 24 also had adjacent normal prostate tissue. To identify metabolites associated with tumor aggressiveness, we obtained matched tumor and normal FFPE tissue from 60 prostate cancer patients (Gleason ≥4+3, n=30; Gleason ≤3+4, n=30) who underwent RP participating in the UNC Health Registry/Cancer Survivorship Cohort. Following log transformation and imputation of missing values with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between groups. Random forest analysis evaluated predictive accuracy of metabolites for distinguishing tumor from normal prostate tissue. We identified 768 compounds of known identity in frozen and 119 in matched FFPE tissues. Therefore, only 15% of the metabolites detected in frozen samples were recovered from corresponding FFPE tissue, with 80% of these common to both FFPE and matched frozen tissues. We found a predictive accuracy of 78% and 67% for frozen and FFPE tissue, respectively, for distinguishing tumor from normal. Of 39 metabolites significantly altered between matched tumor and normal FFPE tissue, 47% belonged to the lipid class, and despite a greater number of significantly altered metabolites in frozen tissue (246), the proportion belonging to the lipid class was similar (50%). Citrate, integral to normal prostate metabolism, and citrulline, involved in nitric oxide synthesis, had significantly lower levels in aggressive versus non-aggressive tumors. Cell membrane phospholipids were found at significantly higher levels in aggressive tumors. Common patterns of altered metabolites in tumor vs. normal prostate were observed irrespective of tissue preservation method, despite loss of a large proportion of metabolites during the process of FFPE. As such, FFPE studies are feasible particularly when lipid metabolism is of interest as this class appears one of the best preserved in FFPE. This work will pave the way for incorporating metabolomics profiling of FFPE specimens into epidemiology and registry-based studies of prostate cancer. Citation Format: Sophia Rachael Halliday, Linnea T. Olsson, Alina Hamilton, Sivapriya Ramamoorthy, Jason Kitchen, Erin Kirk, Laura Farnan, Adrian Gerstel, Melissa A. Troester, Jannette T. Bensen, Sara E. Wobker, Emma Allott. Metabolomics profiling of formalin-fixed paraffin-embedded prostate tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2347.

Expression of miR-127, miR-154, and miR-183 in Medullary Thyroid Carcinoma Tumors.

Background:Medullary thyroid cancer (MTC) accounts for 5%–10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues.Methods:In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR-127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results:Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant.Conclusion:The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC.

Discrimination of Melanoma Using Laser-Induced Breakdown Spectroscopy Conducted on Human Tissue Samples

Discrimination and identification of melanoma (a kind of skin cancer) by using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics methods are reported. The human melanoma and normal tissues are used in the form of formalin-fixed paraffin-embedded (FFPE) blocks as samples. The results demonstrated higher LIBS signal intensities of phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), and calcium (Ca) in melanoma FFPE samples while lower signal intensities in normal FFPE tissue samples. Chemometric methods, artificial neural network (ANN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and partial least square discriminant analysis (PLS-DA) are used to build the classification models. Different preprocessing methods, standard normal variate (SNV), mean-centering, normalization by total area, and autoscaling, were compared. A good performance of the model (sensitivity, specificity, and accuracy) for melanoma and normal FFPE tissues has been achieved by the ANN and PLS-DA models (all were 100%). The results revealed that LIBS combined with chemometric methods for detection and discrimination of human malignancies is a reliable, accurate, and precise technique.

Targeted metabolomics in formalin-fixed paraffin-embedded tissue specimens: Liquid chromatography-tandem mass spectrometry determination of acidic metabolites in cancer research

Formalin-fixed paraffin-embedded (FFPE) tissues play an irreplaceable role in cancer research. Although extensive research has been conducted for the detection of DNA, RNA and proteins in FFPE samples, literature dealing with the FFPE determination of small molecules is scarce. In this study, we aimed to explore the potential of targeted metabolomics in FFPE specimens. For that purpose, we developed a LC-MS/MS method for the quantification of acidic metabolites in FFPE samples. The method involves trimming tissue slices from FFPE blocks, deparaffinization, lysis of the tissue, o-benzyl hydroxylamine derivatization and LC-MS/MS detection. Deparaffinization and lysis steps were optimized to maximize the analytes extraction and to minimize the effect of the ubiquitous presence of some metabolites in the paraffin. Two validation approaches were applied: (i) using blank paraffin as matrix and (ii) using actual human FFPE tissue samples by standard additions. The method quantified 40 metabolites with appropriate accuracy (commonly 80–120%) and precision (CV 2–19%) in both validation approaches. LLOQs ranging 0.88–2001 pg mg−1 with low-moderate matrix effects (commonly 85–115%) were obtained. FFPE samples from 15 patients with colorectal cancer were analyzed and metabolites concentrations in tumor vs matched normal FFPE tissues were compared. Results show that tumor tissues have a well-established fingerprint including an increase in ketogenesis, a decrease in lipogenesis and an imbalance in the tricarboxylic acid cycle.

Phosphorylation Ratio Determination in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue with Targeted Mass Spectrometry.

Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.

Cyclin dependent kinase inhibitor 2A and miR-671-5p expression profile in Iranian glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor which necessitates a search for biomarkers to provide targeted therapies. The dysfunction of microRNA (miRNA) is associated with tumorigenic processes. So, they may be used as potential biomarkers for cancer management. We aim to investigate the expression levels of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) and miR-671-5p in formalin-fixed, paraffin-embedded (FFPE) GBM compared to normal tissues. CDKN2A and miR-671-5p expression levels were evaluated using Real-time RT-PCR in 50 GBM and 50 normal FFPE tissue samples. Our data showed that CDKN2A significantly low expressed and miR-671-5p overexpressed in tumor tissues compared with normal. In addition, a significant negative correlation also found between these gene expressions. Our finding suggests that miR-671-5p may function as a potential oncomiR that directly targets CDKN2A in GBM. Moreover CDKN2A and miR-671-5p could be used as a discriminatory means to categorize subjects as healthy or patient. We observed a significant association between CDKN2A and miR-671-5p expression in GBM, which can be concluded that they have a promising potential to use as primary cancer biomarkers.

1410P - Clinical and analytical accuracy of a 523 gene panel next-generation sequencing (NGS) assay on formalin-fixed paraffin-embedded (FFPE) solid tumour samples

BackgroundWe developed a tumor-only NGS assay (TruSight assay), a 523 gene panel covering 1.94 megabases (Mb), that detects small DNA variants, provides a microsatellite instability (MSI) score, and estimates tumor mutational burden (TMB) from FFPE tissue DNA. We demonstrate the assay’s ability to detect a variety of biomarkers compared to various orthogonal methods across a range of solid tumor tissues. We also demonstrate accuracy in determining TMB status, using a clinically based cutoff, in non-small cell lung cancer (NSCLC) FFPE samples compared to tumor-normal whole exome sequencing (WES). MethodsThe study used ∼400 paired tumor-normal FFPE samples from 7 tumor types, including 240 NSCLC. Tumor samples were tested using the verified assay workflow including software analysis. All tumor and normal FFPE were tested with internal WES (with an independent analysis pipeline) and most with a commercial MSI PCR kit (Promega). The TMB score threshold, in mutations (mut)/Mb, was established for the TruSight assay and WES using 119 independent NSCLC FFPE samples and correlated with an external published cutoff. ResultsAcross different tumor types, there was high concordance in small DNA variant calling (>95% positive percent agreement [PPA], >99% negative percent agreement [NPA]) between the TruSight assay and WES. For MSI, >95% PPA and 100% NPA were achieved between the TruSight assay and MSI-PCR. TMB scores from the TruSight assay showed good correlation with WES TMB scores (R2 >0.95 on linear regression). Among the NSCLC samples, TMB scores (range 0 – 184.6 mut/Mb) determined by the TruSight assay correlated well with WES (R2 0.94). Using cutoffs of 12.3 and 8.3 mut/Mb (TruSight assay and WES, respectively) to assign TMB High and Low statuses, overall percent agreement (OPA) was 85%. ConclusionsThe tumor-only TruSight assay showed high accuracy in detecting biomarkers across a range of solid tumors. The assay showed a good correlation in TMB score and agreement in TMB status with tumor-normal WES in a collection of NSCLC samples. Legal entity responsible for the studyThe authors. FundingIllumina. DisclosureI. Deras: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. T. Du: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Zhao: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. N. Haseley: Full / Part-time employment: Illumina. A. Yazdanparast: Full / Part-time employment: Illumina. T. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. A. Mentzer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. A. Purdy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. B. Crain: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Echegaray: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. D. Lee: Full / Part-time employment: Illumina. J. Lee: Full / Part-time employment: Illumina. J. Silhavy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. K. O’Brien: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Vijayaraghavan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Garcia: Full / Part-time employment: Illumina. R. Haigis: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. J. Dockter: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina.

Nonfouling, Encoded Hydrogel Microparticles for Multiplex MicroRNA Profiling Directly from Formalin-Fixed, Paraffin-Embedded Tissue.

MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.

Whole exome sequencing in the rat

BackgroundThe rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants.ResultsInformatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes.ConclusionsAn in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.

FFPE breast tumour blocks provide reliable sources of both germline and malignant DNA for investigation of genetic determinants of individual tumour responses to treatment

BackgroundBio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood.MethodsExome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively.ResultsOverlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson’s correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94.ConclusionsThere is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material.

Abstract 3388: Copy number alteration in primary melanoma

Abstract The systematic analysis of the genomic characteristics of melanoma primaries has been infeasible given the limited size of these formalin-fixed paraffin embedded (FFPE) lesions. Further studying a clearly ascertained, deeply phenotyped patient population allows meaningful extrapolation of the prevalence of distinct genomic features and investigation of the association of these features with lifestyle exposures and germline profiles. For these reasons, we focused on a recently recruited patient cohort, the Leeds Melanoma Cohort (LMC) consisting of 2184 recruits from a distinct geographical region of Northern England; the only selection applied to recruitment was to only include those whose melanoma was less than 0.75 mm Breslow thickness in the early years of recruitment. Tumor samples from 703 cases have been analyzed for genome-wide gene expression (Pozniak et al, this meeting). For this study, we wanted to explore the prevalence of copy number alterations (CNA) and the mutation profile of tumors (reflecting the association of C&amp;gt;T mutations with UV exposure, the primary environmental risk factor) and so we have adopted a next-generation sequencing (NGS) approach. Tumor blocks were recovered from local pathology laboratories for those cases already deceased at the time that this CNA analysis was initiated plus those surviving more than 5 years post diagnosis at this same time. Blocks at risk of being compromised for clinical purposes by sampling for this research study were excluded from sampling; this amounted to almost 50% of identified samples. A total of 333 NGS libraries were sequenced on an Illumina GAII or HiSeq sequencer to produce &amp;gt;100bp paired-end reads (either 5 or 1 per lane). DNA reads were aligned and mapped achieving approximately 1.8x coverage (9.4x for those at 1 per lane); the number of reads falling within each 10kb window was adjusted for GC content and mappability and compared to a composite normal FFPE sample to determine local copy number. We adopted various approaches to assess the informativeness of the methodology. (i) Replicate samples showed high reproducibility (all p &amp;lt; 0.0001). (ii) Focused analysis of the CDKN2A region with segmentation analyses to determine CNA breakpoints yielded high quality, biologically plausible data revealing multiple copy number events. 67% of samples showed no CNA across the 4 Mb region covering CDKN2A while, as expected, only a small proportion of CNAs did not involve CDKN2A. Four common distinct patterns were observed. Copy number loss ranged from 20kb to &amp;gt; 4 Mb. (iii) A germline CNA could reliably be identified from the tumor data. (iv) CDKN2A expression levels correlated with estimated copy number. We have shown that small FFPE melanomas can be characterized for genomic alterations using NGS opening up the potential for studies associating epidemiological and germline profile with the resulting tumor. 1. Newton-Bishop, J.A., et al. (2009) J Clin Oncol, 27, 5439-5444. Funded by Worldwide Cancer Research and Cancer Research UK. Citation Format: D. Timothy Bishop, Anastasia Filia, Alastair Droop, Joey Diaz, Mark Harland, Jon Laye, Juliette Randerson-Moor, Julia A. Newton-Bishop. Copy number alteration in primary melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2017-3388

Abstract 4229: Physical expansion of tissue microarrays for high-resolution imaging of normal and cancer samples with conventional microscopy

Abstract BACKGROUND Archival formalin fixed paraffin embedded (FFPE) tissues are an excellent and abundant source of human cancer and normal tissues, useful for cancer research and diagnosis. Moreover, FFPE tissue microarrays enable analysis of hundreds of samples from many different patients on the same slide. Super resolution microscopes have been used for cancer tissue imaging; however, these methods are expensive and require long recording times, limiting their usefulness for the cancer research community. Recently, a new approach (Expansion Microscopy) was developed to enable physical magnification and high resolution imaging of cell lines and fresh-frozen (and fixed) mouse brain with conventional microscopes (Chen F, Tillberg PW, Boyden ES, 2015, Science 347:543-548). In the current study, we developed expansion microscopy for expanding and imaging formalin fixed paraffin embedded normal and cancer human tissues, both in tissue microarrays and whole tissue slides. METHODS Expansion microscopy (ExM) physically magnifies tissue samples by embedding them in a dense swellable polymer, anchoring key biomolecules to the polymer mesh, and adding water to swell the polymer. ExM physically magnifies the brain tissue with nanoscale isotropy and a post-expansion measurement error of 2-4%. Proteins can also be visualized by a modified immunofluorescence assay (Chen F et al., Science, 2015). In the present study, we optimized the ExM chemistry, labeling, and imaging methodologies to enable ExM to be used in cancer research and diagnosis, for morphological and protein imaging and analysis of both tissue microarrays and whole tissue slides on a wide variety of human tissues. Nuclei were detected by DAPI, and protein markers, including those of stroma (vimentin) and epithelium (keratins), were detected using immunofluorescence. RESULTS and CONCLUSIONS We developed ExM protocols to enable expansion of human normal and cancer tissues ∼4.5x in linear dimension, with a post-expansion measurement error of 5% or less. We successfully expanded normal and cancer human FFPE tissue microarrays containing over eight different tissue origins, including breast, prostate, lung, colon, pancreas and ovary. We expanded fresh frozen tissue samples of normal and cancer tissues. Our method makes possible the use of ExM on routinely collected FFPE pathology samples, enabling super resolution optical investigation of morphology and protein expression/localization in tissue microarrays with conventional fluorescent microscopy. Future applications include both large-scale retrospective and prospective studies of carcinogenesis in clinical tissue samples. * Yongxin Zhao and Octavian Bucur contributed equally; # Edward Boyden (esb@media.mit.edu) and Andrew H. Beck are corresponding authors (abeck2@bidmc.harvard.edu); Citation Format: Octavian Bucur, Yongxin Zhao, Edward Boyden, Andrew H. Beck. Physical expansion of tissue microarrays for high-resolution imaging of normal and cancer samples with conventional microscopy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4229.

Abstract 52: QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting

Abstract The emerging field of precision medicine considers the patient's individual genetic make up, environment, and lifestyle to arrive at individualized treatment. For cancer patients, analysis of the tumour genome can reveal actionable alterations, including mutations (e.g., BRAF mutation in melanoma), translocations (e.g., t(9;22)(q34;q11) in CML) and copy number changes (e.g., HER2 amplification in breast cancer). Formalin-fixed paraffin embedded (FFPE) biopsy or surgical pathology samples are often the only materials available for analysis, highlighting the need for genomic analysis techniques that perform reliably with such materials. We present a complete pipeline for DNA copy number profiling by sequencing called quantitative DNA sequencing (QDNAseq), which offers a cost effective procedure for genome-wide DNA copy number analysis, facilitates correct biological interpretations from samples with a varying range of quality and is suitable for routine pathology diagnostics from fresh, frozen and FFPE materials. QDNAseq is implemented in R and made available through BioConductor and Galaxy. The pipeline requires as little as ∼0.1× genome coverage (∼6 million single-end 50nt reads per sample) and utilizes a depth of coverage (DOC) approach. A two-dimensional LOESS correction is applied for GC content and mappability and data from the 1000 genomes project are used to compile a blacklist of problematic genome regions. Downstream analyses have been included in QDNAseq by adapting algorithms previously developed for segmentation (DNAcopy) and the calling of gains and losses (CGHcall). QDNAseq is able to produce profiles with noise levels near the probabilistic lower limit imposed by read counting and yields a higher resolution than is available from microarrays. Some samples, however, have variances clearly above the probabilistic lower limit and are characterized by a wave pattern. This pattern originates in the sample, as it is reproducible, both in magnitude and in shape along the genome. Much of this wave bias can be reduced by regression with profiles from matched normal FFPE DNA samples using the algorithm implemented in NoWaves. The QDNAseq procedure is highly cost effective and robust. We have processed over 1500 clinical samples obtained from the FFPE archives of more than 25 institutions from Europe and North America. Citation Format: Daoud Sie, Ilari Scheinin, Stef Lieshout, van, Martijn Cordes, Daniel Pinkel, Donna G. Albertson, Mark A. Wiel, van de, Bauke Ylstra. QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 52.

Abstract 3269: QTLs in breast tumor and breast normal adjacent FFPE specimens from the Nurses’ Health Study

Abstract Rationale: Genome-wide association studies (GWAS) of breast cancer have identified 71 risk alleles, majority of which are located in intergenic or intronic regions. We used a network medicine approach to associate breast cancer risk single nucleotide polymorphisms (SNPs) with transcript abundance in formalin fixed paraffin embedded (FFPE) breast tissue. Identification of expression quantitative loci (eQTLs) may help to better understand the regulatory mechanisms by which these risk variants influence breast cancer susceptibility. Method: We identified invasive postmenopausal breast cancer cases in the Nurses’ Health Study (NHS) diagnosed from 1990-2004 with GWAS data and sufficient RNA for expression profiling in breast tumor and normal adjacent breast tissue. RNA was extracted using the Qiagen AllPrep RNA isolation kit, amplified using the NuGen Ovation FFPE WTA kit, and profiled using the Affymetrix Human Transcriptome Array (HTA 3.0v1). The HTA includes 6,892,960 features for measuring gene expression, alternative splicing, coding SNPs, and noncoding transcripts. After filtering and removing probes with a low dynamic range in our samples, we included 25,000 gene expression probes in our QTL analyses. We mapped these probes to genes using hg19. Results: Analyses were conducted separately for 376 breast tumor and 274 normal adjacent samples. We conducted quality control analyses, corrected for assay plate-to-plate variation and included patient's age at diagnosis and year of diagnosis as covariates in the multivariate linear regression model. We identified 11 trans eQTLs in normal adjacent breast tissue, 11 trans eQTLs in estrogen receptor (ER)+ breast tumor samples, and 12 trans eQTLs in ER- breast tumor samples (permutation adjusted p-value&amp;lt;0.05). We also developed a new method, functional quantitative trait loci (fQTL) analysis, to gain additional pathway insight into genetic associations important in breast cancer. In the fQTL analysis we tested for the association between SNPs and the expression of gene functional classes and pathways, evaluating the hypothesis that SNPs may also be associated with regulation of processes in addition to individual genes. Using a cutoff of false discovery rate &amp;lt;10%, we identified 2 SNPs associated with 2 Gene Ontology Molecular Functions in normal adjacent breast tissue, 1 SNP associated with 5 Molecular Functions in ER- tumor samples but no significant SNP associations in ER+ tumor samples. Integrated eQTL and fQTL analyses and variant annotation are ongoing. Conclusion: In summary, our results provide functional insights on the underlying biology of loci identified in breast cancer GWAS in the specimen type that is most impactful in translation to clinical practice. Identification of gene transcripts that can be measured in FFPE tissue and are associated with breast cancer risk loci is critical in understanding the mechanism by which these variants affect risk and mediate disease processes. Citation Format: Alejandro Quiroz-Zarate, Benjamin J. Harshfield, Rong Hu, Nick Knoblauch, Andrew H. Beck, Vincent Carey, Susan E. Hankinson, Rulla M. Tamimi, David J. Hunter, John Quackenbush, Aditi Hazra. QTLs in breast tumor and breast normal adjacent FFPE specimens from the Nurses’ Health Study. [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 3269. doi:10.1158/1538-7445.AM2014-3269