For Formalin-fixed Paraffin-embedded Samples Research Articles

Characterizing the tumor immune microenvironment of ependymomas using targeted gene expression profiles and RNA sequencing

BackgroundDefining the tumor immune microenvironment (TIME) of patients using transcriptome analysis is gaining more popularity. Here, we examined and discussed the pros and cons of using RNA sequencing for fresh frozen samples and targeted gene expression immune profiles (NanoString) for formalin-fixed, paraffin-embedded (FFPE) samples to characterize the TIME of ependymoma samples.ResultsOur results showed a stable expression of the 40 housekeeping genes throughout all samples. The Pearson correlation of the endogenous genes was high. To define the TIME, we first checked the expression of the PTPRC gene, known as CD45, and found it was above the detection limit in all samples by both techniques. T cells were identified consistently using the two types of data. In addition, both techniques showed that the immune landscape was heterogeneous in the 6 ependymoma samples used for this study.ConclusionsThe low-abundant genes were detected in higher quantities using the NanoString technique, even when FFPE samples were used. RNA sequencing is better suited for biomarker discovery, fusion gene detection, and getting a broader overview of the TIME. The technique that was used to measure the samples had a considerable effect on the type of immune cells that were identified. The limited number of tumor-infiltrating immune cells compared to the high density of tumor cells in ependymoma can limit the sensitivity of RNA expression techniques regarding the identification of the infiltrating immune cells.

Abstract 2944: Rapid and accurate variant calling of FFPE samples with the Genexus System

Abstract Next-Generation Sequencing technology has enhanced oncology research by enabling the detection of all cancer related variants into one assay for research and drug discovery programs. The Oncomine࣪ Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent࣪ Genexus࣪ System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated sample to result workflow in 30hrs. This study demonstrates ≥ 95% Sensitivity and PPV for detecting SNV, Indel, and Copy Number variants of clinical FFPE samples along with fusions. When tested using analytical controls, the Genexus System achieves ≥ 98% Sensitivity for hotspot variants, ≥ 95% for de novo variants, and 100% Sensitivity for fusion variants. The Ion Torrent࣪ Genexus࣪ System is fully automated and consists of two software linked instruments, the Ion Torrent࣪ Genexus࣪ Purification System and the Ion Torrent࣪ Genexus࣪ Integrated Sequencer. For this study, the purification system was used to sequentially extract and quantify DNA & RNA samples from human colon and lung FFPE tumor tissue. The purification instrument provides minimal hands-on-time, ease of use and compatibility with the integrated sequencer. It extracts and quantifies the nucleic acids, records the quantitation values and transfers the nucleic acids to an output plate. The plate is transferred from the purification instrument directly to the sequencer for sample dilution, library preparation, and sequencing using the Oncomine࣪ Comprehensive Assay v3 panel. Variant calling analysis of the DNA & RNA pairs is completed immediately following sequencing. Results show that the variants are detected reproducibly with ≥ 95% Sensitivity and PPV when compared to the Ion GeneStudio࣪ S5 System as an orthogonal method. All variants in the report were correct with p-values ≤ 10-5. Additionally, a subset of the extracted samples was evaluated using Sanger Sequencing to verify hotspot mutations found in samples sequenced on both the Genexus࣪ System and GeneStudio࣪ S5 System. Results show that all expected hotspot variants were detected. In summary, the Ion Torrent࣪ Genexus࣪ System is a reliable and fast turnaround solution for sample-to-variant calling results. When used with the Oncomine࣪ Comprehensive Assay v3 panel, the system provides accurate identification of tumor markers for oncology research. Citation Format: Iris Casuga, Frances Chan, Milton Huynh, Gregory R. Govoni, Kayla Zochowski, Thilanka Jayaweera, Janice Au-Young. Rapid and accurate variant calling of FFPE samples with the Genexus System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2944.

Abstract 2282: Rapid and accurate variant calling of FFPE samples with the genexus system

Abstract Next Generation Sequencing technology has enhanced oncology research by enabling the detection of relevant variants for clinical and drug discovery programs. The Oncomine Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent Genexus System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated workflow from sample to result in 30hrs. This study demonstrates ≥ 90% accuracy for detecting SNV/Indel/CNV and Fusion variants. The Genexus System consists of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The purification system is used first to extract and quantify 6 FFPE sections from human colon and lung tissue for matching DNA & RNA pairs. The nucleic acid output plate from the purification instrument is directly transferred to the sequencer for library preparation and sequencing using the Oncomine Comprehensive Assay v3. Variant calling analysis of 6 DNA & RNA pairs are completed the following day. Results show that the variants were detected with ≥ 90% Sensitivity when compared to the Ion Torrent GeneStudio S5 System as an orthogonal method. All variants report p-values ≤ 10^-5 signifying more confidence that the variant call is correct. The Genexus System is a seamless solution for sample to variant calling results. Citation Format: Iris Casuga, Jason Gioia, Janice Au-Young, Frances Chan, Milton Huynh, Michael Crowe. Rapid and accurate variant calling of FFPE samples with the genexus system [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 2282.

Technical validation of a high-sensitivity target capture NGS assay using unique molecular identifier approach.

e13657 Background: Identification of a broad spectrum of somatic mutations is crucial to guide targeted therapy such as for non-small cell lung cancer (NSCLC) patients. In the clinical environment, it requires well validated NGS workflow both for the wet-lab and dry-lab procedures. Here we describe a high sensitivity target NGS assay to accurately capture single nucleotide variants (SNVs), short insertions and deletions (indels), copy number alterations and gene rearrangements for formalin-fixed paraffin-embedded (FFPE) NSCLC patient samples. Extensive analytical validation was performed following the checklists of College of American Pathologists. Methods: Next generation sequencing (NGS) libraries were prepared using extracted DNA from FFPE tissue NSCLC patient samples. The protocol for library generation was optimized in several steps and incorporated 10bp unique molecular identifiers (UMIs). The libraries were sequenced on Illumina HiSeq X-Ten platform. The sequence data was analyzed by an in-house bioinformatics pipeline to call somatic mutations at an average depth of 4000X. Results: We tested the accuracy of 68 clinical tumor samples that were also validated by conventional or alternative methods in the third party CAP accredited labs. We observed 100% sensitivity and 100% specificity compared with the other lab¡¯s validation results. To define the limit of detection (LOD) for different mutation types, clinical DNA samples containing different variants were diluted with normal DNA. The LODs for SNV (as in EGFR L858R) and indel (as in EGFR 19del) were 0.5% and 1%, respectively. Addressing the LOD of fusion and copy number alteration is usually challenging. Our NGS assay was able to achieve 2% LOD for gene rearrangement (fusion) and 3.5 copies for copy number amplification. The high reproducibility was also achieved by inter- and intra- replicate experiments. Our NGS assay showed better performance than other widely used commercial NGS assay panels. Conclusions: We have validated an NGS based approach with UMI technology that is able to achieve high accuracy and sensitivity as low as 0.5% for detection of somatic mutations, which will improve the clinical testing performance for NSCLC FFPE samples with low allele frequencies of driver mutations.

Abstract 3833: Systematic evaluation of transcriptome sequencing shows comparable profiles for an exome-capture method for formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues and the standard poly-A method for matched fresh frozen (FF) tissues

Abstract Background: Tissue samples are routinely preserved as FFPE blocks. This abundant sample reservoir holds promises in clinical omics for disease diagnosis and precision cancer medicine. However, due to extensive cross-linking, nucleic acids are not efficiently extracted from FFPE samples and are heavily degraded, posing challenges for molecular assays including next generation sequencing. Our study aims to identify the RNA sequencing (RNA-seq) library prep method for FFPE samples that provides comparable data to the established poly-A enrichment method for FF tissue. Methods: RNA-seq data were collected on matched FF and FFPE samples from triple negative breast cancer (TNBC; n=7) and normal breast tissues (n=2). Tissues were sourced from a commercial vendor. Three library preparation protocols from Illumina were tested: poly(A) enrichment protocol (Poly-A; FF only), ribosomal RNA deletion protocol (Ribo-Zero Gold), and exome-capture based protocol (RNA-Access). Data were compared on absolute and differential gene expression, as well as molecular subtyping for breast cancer (PAM50; Parker 2009). Results: Both absolute expression values and log fold changes between tumor and normal samples correlate strongly across different tissue preservation and library preparation methods. Among tested methods, RNA-Access had the highest yield of mappable reads on coding region and provided uniform transcript coverage with less rRNA contamination. Regardless of the protocol, all TNBC samples were classified as PAM50 basal-like subtype. As expected, FF with either Ribo-Zero or RNA-Access had the strongest correlation with FF PolyA on the distance to subtype centroids (r=0.99). For FFPE methods, RNA-Access (r=0.94) had the strongest correlation with FF PolyA, followed by Ribo-Zero (r=0.85). Principal component analysis showed that samples were primarily segregated by technical differences (library preparation or tissue preservation method) rather than biological differences (tumor/normal status or patient identification), suggesting strong technical bias in the data. Gene families and biological processes underlying the differentially expressed genes due to technical variations included ribosomal, histone and metabolic pathways proteins. The aforementioned technical bias can be largely eliminated by a batch effect adjustment method (COMBAT; Johnson 2007). Conclusion: As expected, RNA-seq data is primarily impacted by technical differences. RNA-Access based transcriptome sequencing on breast FFPE samples generated highly concordant gene expression profiles when compared to data derived from FF samples. Our study supports the use of RNA-Access based transcriptome sequencing on FFPE samples when FF samples are not available. Citation Format: Xi Zhao, Marianna Zavodovskaya, Luting Zhuo, Kevin Kwei, Xin Guo, Zhaoshi Jiang, Scott D. Patterson, Carrie B. Brachmann. Systematic evaluation of transcriptome sequencing shows comparable profiles for an exome-capture method for formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues and the standard poly-A method for matched fresh frozen (FF) tissues [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 3833. doi:10.1158/1538-7445.AM2017-3833

Abstract 3585: Determination of gene amplifications with a next-generation sequencing cancer panel

Abstract TruSight® Tumor 15* (TST15) uses next-generation sequencing technology to detect somatic point mutations, insertions, and deletions in 15 genes that are commonly mutated in solid tumors. It accurately detects low-frequency mutations with low DNA input and is optimized for formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The test is a multiplexed PCR panel that includes primers to support amplification detection in the EGFR, ERBB2, and MET genes. Here we describe development of a bioinformatics analysis method to enable detection of these gene amplifications. DNA extracted from 31 FFPE samples with amplifications in EGFR, ERBB2, or MET—some with dual amplifications—were diluted with DNA extracted from 18 normal FFPE samples to create a total of 67 unique samples at low-level amplification. Undiluted and diluted samples were assessed for quality based on amplifiability (ΔCq or library yield) and tested with TST15. For comparison, the samples were also tested with droplet digital PCR (ddPCR) to determine the copy ratio of target to wild type. For amplifiable samples, the TST15 algorithm was able to detect amplifications in EGFR and ERBB2 at ddPCR ratios of 1.4 and 1.6, respectively. ddPCR was unable to accurately quantify low MET amplifications; therefore, expected ddPCR ratios were used for comparison. The TST15 algorithm was able to detect amplifications in MET samples at an expected ddPCR ratio of 1.3. Poor sample quality (i.e., high ΔCq or low library yield) impaired amplification detection. In some samples, the presence of multiple amplifications also impaired detection. The addition of this algorithm to the TST15 workflow will allow researchers to more accurately characterize tumor samples by detecting somatic mutations and amplifications simultaneously. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ina L. Deras, Aaron Wise, Chen Zhao, Christine Glidewell-Kenney, Phillip Le, Elizabeth Upsall Aderhold, Karen Gutekunst. Determination of gene amplifications with a next-generation sequencing cancer panel [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 3585. doi:10.1158/1538-7445.AM2017-3585

Abstract 5409: Assessment of the MammaPrint 70-gene profile using RNA sequencing technology

Abstract Introduction: Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Recently, MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, was successfully translated to FFPE tissue showing to be substantially equivalent to fresh tissue. In recent years, RNA-sequencing (RNA-Seq) became the standard method for transcriptome analysis, because of its low background signal and its ability of quantifying a large dynamic range of expression levels. Here we report a preliminary analysis of the FFPE MammaPrint 70-gene profile using RNA-Seq technology and the comparison with the MammaPrint® microarray diagnostic test in a series of FFPE samples. Methods: RNA-Seq was carried out using a strand-specific RNA library preparation followed by target enrichment of the coding region of the human transcriptome without relying on the presence of poly-A tail. RNA sequencing libraries were prepared starting from a minimal amount of 20 ng of total RNA based on the DV200 metric assessment. The library pools were single-end sequenced on the Illumina HiSeq 2500 instrument at the length of 65bp. The resulting sequences were mapped to the human reference genome (build 38) using TopHat v2.1. Tophat was guided by using a transcriptome index from Ensembl (version 77). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Gene expressions were normalized with Count Per Million (CPM) normalization and log2 transformed afterwards. Microarray data of the sample were available for analysis comparison. Results: On average, we obtained 22 million reads assigned to gene per sample (min=15M, max=28M). The number of reads assigned to genes vary from 61% to 70% of the total number of reads. Between 80% and 90% of the reads assigned to genes mapped to protein coding genes which is comparable to fresh frozen material. The 70-gene signature was successfully mapped to the RNA-Seq genes. A median raw read-count of 384 was observed for the 70-gene profile among the samples. Importantly, we observed a high concordance (R2 Pearson correlation=0.97) between the MammaPrint index calculated using the RNA-Seq data and the correspondent Microarray MammaPrint index. Additionally, the BluePrint profile, a microarray diagnostic test for breast cancer molecular subtyping, was successfully translated to the RNA-Seq platform. As with the MammaPrint profile, BluePrint showed high concordance between the two technologies with high correlation values for each of the subtypes (Luminal R2 Pearson correlation=0.98, Basal R2 Pearson correlation=0.97, HER2 R2 Pearson correlation=0.77). Conclusions: Next Generation RNA-sequencing is a feasible technology to assess diagnostic signatures, such as the 70 gene MammaPrint and BluePrint profiles. Citation Format: Lorenza Mittempergher, Jacob B. Spangler, Mireille H. Snel, Leonie J. Delahaye, Iris de Rink, Sun Tian, Annuska M. Glas, Rene Bernards. Assessment of the MammaPrint 70-gene profile using RNA sequencing technology [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 5409. doi:10.1158/1538-7445.AM2017-5409

Development of a pan-cancer 15 gene expression signature to detect a subgroup driven by EMT/MAPK signalling.

11616 Background: Epithelial–mesenchymal transition (EMT) is the conversion of epithelial cells to mesenchymal cells and involves loss of cell–cell adhesion and cell polarity and increased motility, invasiveness and metastases. The EMT process has therefore been under investigation as a new target for anticancer drug discovery. The aim of this work was to develop an EMT biomarker suitable for formalin-fixed paraffin embedded (FFPE) tissue that could be used for patient treatment selection. Methods: Unsupervised hierarchical clustering of ovarian cancer gene expression data (TCGA. 2011) previously identified an EMT subgroup. We confirmed this EMT subgroup in FFPE tissue using 265 high grade serous ovarian cancer (HGSOC) FFPE samples. The analysis was extended to show existence of an EMT subgroup across a range of solid tumours including colon, lung, melanoma and prostate cancer. Further to this, a common gene list was generated to include only transcripts with high variability and expression across diseases, and used as a starting list for development of a 15 transcript assay which can be used to prospectively identify the EMT subgroup from archived tissue. Results: The 15 gene expression assay was a poor prognostic marker in Colorectal, (Relapse free survival: HR = 1.46 [95% CI:1.07-1.98]); Lung, (Relapse free survival: HR = 2.18 [95% CI:1.33-3.56]); Prostate cancer, (Biochemical recurrence: HR 2.49 CI: 1.43-4.34) and was associated with activated MAPK (phospho-MAPK) in preclinical models and clinical samples (p < 0.05). The assay score was reduced by MEK inhibition (p < 0.05) and elevated by KRAS, NRAS and MEK1 overexpression (p < 0.05). The assay predicted response to the MEK inhibitors Trametinib and Selumetinib in cell line models (p < 0.001). Conclusions: A 15 gene expression assay has been developed from FFPE samples across multiple diseases to detect an EMT molecular subgroup associated with MAPK signalling. The assay predicted sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the assay as a predictive biomarker in clinical samples from patients treated with EMT or MEK targeted therapies.

Human epidermal growth factor receptor 2 amplification detection by droplet digital polymerase chain reaction in formalin-fixed paraffin-embedded breast and gastric cancer samples.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for the precise individualized treatment including trastuzumab of HER2-positive breast and gastric cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the routine analyses for formalin-fixed paraffin-embedded (FFPE) samples. However, IHC is variable and depends on the evaluator, and FISH is a labor intensive and expensive method. We evaluated the feasibility of droplet digital polymerase chain reaction (ddPCR) as a precise and quantitative method for HER2 amplification test. We used ddPCR to confirm HER2 amplification status in 24 breast cancer and 29 gastric cancer samples to validate the HER2 cutoff value in ddPCR. After setting cutoff value, all the above-mentioned samples were tested by IHC. Afterward, another 51 equivocal IHC 2+ gastric cancer samples were further determined by FISH and ddPCR, respectively, and the concordance between ddPCR and FISH was calculated. We set the HER2 cutoff value at 1.8. The concordance rate of HER2 status between ddPCR and IHC was 94.4% (17 out of 18) in 24 breast cancer samples. In 29 gastric cancer specimens, the concordance rate of HER2 amplification between ddPCR and IHC was 100% (22 out of 22). At last, compared with FISH determined HER2 status, ddPCR HER2 scores correctly classified 44 of 51 cases with 86.3% concordance in 51 equivocal IHC 2+ gastric cancer samples. ddPCR was able to identify HER2 amplification status in breast and gastric cancers with precise correlation with IHC and FISH results. This method might become a standard method for testing FFPE samples. However, the technology requires further research.

Primary resistance to Hepatitis B Virus in the treatment-naive Patients

No: 1714 Presentation at ESCV 2015: Poster 2 Primary resistance to Hepatitis B Virus in the treatment-naive Patients M. Altindis1,∗, L. Demir1, I. Uslan2, Ozdemir Mehmet3, M. Baykan3, M. Kosecik4 1 Sakarya University School of Medicine, Dept of Clinical Microbiology, Sakarya, Turkey 2 Sakarya University School of Medicine, Dept of Gastroenterology, Sakarya, Turkey 3 Necmettin Erbakan Uni School of Medicine, Dept of clinical Microbiology, Konya, Turkey 4 Sakarya University School of Medicine, Dept of Pediatry, Sakarya, Turkey Background:Resistance to nucleos(t)ide analogues (NUC) is the main cause of NUC treatment failures leading to disease aggravation and complications. Our aim was to assess the prevalence of primary resistance to NUCs in a large cohort of treatment-naive patients infected with HBV by means of a highly sensitive method for the detection of minority viral populations. Methods: In this study, presence of resistance mutations in treatmentnaive individuals with chronic hepatitis B (CHB) was investigated in Sakarya University Hospital. A total of 59 patients (24 female, 35 male; age range: 19–66 years) diagnosed as CHB were enrolled in the study with informed consent from Gastroenterology, Sakarya anD Konya. All of the patients were negative for hepatitis C andD viruses and human immunodeficiency virus coinfections, and none had a history of interferon or NA treatment. HBV viral load(Montania® 4896, Bosphore® HBV Quantification Kit, Anatolia), HBV markers and hepatic enzymes in patients were determined via standardized commercial assays. For the detection of NA resistance mutations, a partial sequence of approximately 250 nucleotides, harboring the frequently-observed sites for NA resistance was amplified via nested PCR and characterized by direct sequencing of the amplicons(Bosphore® HBV Drug ResistanceMutation Sequencing Kit, Anatolia and Beckman Coulter CEQ 8000 Genetic Analysis System). The sequences were handled and interpreted for the presence ofmutations via various softwares and a web-based virtual phenotyping tool(CEQ 8000 Genetic Analysis System Version 9). Results: All circulating HBV strains were observed as genotype D(D1, D2, D3). At baseline, 59 patients harbored HBV resistanceassociated variants (RAV), generally in low proportions (0.3–20%). 26.9%, 9.7%, 1.6%, 1.6% of the patients displayed RAVs representing less than 1%, 1–5%, 5–10%, 10–20% of the viral quasispecies, respectively. 3 patients harbored RAVs that represented more than 20% of the quasispecies, including rtq215s, rtl91 and rtQ149K variant at 25%. None of the patients were found to harbor variants with both resistance and fitness mutations, which are generally selected on treatment at the sensitivity level of the technique. Conclusions: Primary HBV resistance to NUCs is frequent in treatment-naivepatients. These results further emphasize theneed for using first-line therapies including NUCs with a high barrier to resistance. http://dx.doi.org/10.1016/j.jcv.2015.07.273 Abstract No: 1725 Presentation at ESCV 2015: Poster 2 Development of an automated extraction system for Formalin-Fixed Paraffin Embedded (FFPE) tissue for the downstream detection of Human Papillomavirus (HPV) D. Guerendiain ∗, C. Moore, K. Cuschieri SHPVRL, NHS Lothian, Edinburgh, UK Background: Characterisation of FFPE biopsy material for HPV status is important for both epidemiological purposes and increasingly, for annotation of oropharyngeal squamous cell cancer (OPSCC). While manual extraction procedures for this application have been published–there is a lack of data on the development and performance of automated systems. The objective of this study was to develop and evaluate an automated extraction system for the detection of HPV nucleic acid using the NucliSENS® easyMAG®. Methods: A total of 60 samples were assessed: 30 FFPE sections related to high grade cervical intraepithelial neoplasia (CIN) lesions and 30 sections were derived from OPSCC. All samples had been extracted previously with manual silica columns (QIAamp DNA Mini Kit) and tested with the Optiplex HPV DNA assay (Diamex). Curled sections were obtained from each block with10 m thickness. Sampleswere extracted using the easyMAG® using a protocol adapted for FFPE material. DNA was tested using the Nanodrop® to evaluate the quantity and purity of DNA extracted. Agreement between the original HPV result associated with the manual extract and the results associatedwith the automated extract were assessed. Results: Nucleic acid yields ranged from 4.8 to 88ng/ l on extractsobtained fromtheeasyMAG® platform.At leastpartial concordance was observed in 93% in both cervical and OPSCC samples with an overall Kappa for HPV detection in the total sample (n=60) of 0.8049 (95% CI: 0.62–0.98). Specific kappas on HPV type-specific agreement will be presented. Conclusion:Our results indicate a high agreement between the easyMAG® and theQIAampDNAMiniKit® for the extractionofHPV DNA in FFPE samples derived from the oropharynx and the cervix This extraction method will reduce hands-on time – particularly for batch extraction. http://dx.doi.org/10.1016/j.jcv.2015.07.274

Abstract A55: KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition

Abstract Background: Given the prevelance of chromosomal instability and gene amplification (amp+) as driver mechanisms in gastroesophageal adenocarcinoma (GEC), we assessed the incidence of KRAS gene amp+ and gene copy number (GCN) along with protein expression, at baseline and after molecular targeted therapy in GEC cell lines/tissues. KRAS undergoes activating mutation in various cancers. KRAS mutation is rare (<5%) in GEC. KRAS is a downstream effector of HER2 and MET tyrosine kinases, and KRAS upregulation may be a resistance mechanism to targeted anti-HER2/anti-MET therapy. We assessed KRAS amp+ in a series of 81 GEC samples and 19 GEC cell lines. We established a KRAS amp+ model to evaluate potential therapeutic strategies. Methods: 81 GEC samples and 19 GEC cell lines were assessed for KRAS GCN by fluorescence in situ hybridization (FISH) using HER2 scoring criteria. KRAS expression was assessed by immunohistochemistry (IHC) and selected reaction monitoring mass spectrometry (SRM-MS) using a novel mulit-plex (GEC-plex) assay for formalin fixed paraffin embedded (FFPE) samples. Patient samples with HER2+ status treated with trastuzuamb as well as a cohort of patients treated with anti-MET therapy were available for pre/post treatment assessment. A KRAS-amplified cell line (Cat2) was established from ascites fluid and grown in vitro and screened for KRAS mutation using deep next-gerenation sequencing to determine if KRAS mutant alleles were present. Tumorigenic activity of Cat2, and another amplified line, MKN1, were assessed using standard proliferation assays in vitro, and an intraperitoneal (IP) murine model assessing time to ascites development (TTA), with/without anti-MEK, anti-PI3K/mTOR, or combinations thereof. Cat2 and MKN1 were also treated with KRAS siRNA and compared to siRNA control in vitro. Results: Assessment of KRAS GCN revealed clustered wild type gene amp+ in 16% (13/81) of FFPE samples, while KRAS mutation was observed in 7% (6/81) (mutually exclusive). KRAS amp+ was found in newly diagnosed metastatic patients (12/55, 22%) more than locally advanced (1/26, 4%) (p=0.06). GCN correlated with expression as determined by IHC and the MS GEC-plex. Patients treated with anti-HER2 or anti-MET therapy were observed to have developed KRAS amp+ in post-treatment biopsies at progressive disease as compared to pre-treatment biopsy. In the cell line, Cat2, KRAS was amp+ (by FISH, mean copies/nucleus: 43.2) and extremely overexpressed by IHC and MS (60 fold over KRAS non-amp+ samples); KRAS was not mutated. In vitro, KRAS siRNA substantially inhibited KRAS amp+ lines versus siRNA control (>80%). We observed diminished KRAS amp+ growth/proliferation both in vitro and in vivo using combined MEK and AKT pathway inhibitors over either alone, and compared to various inhibitor controls (anti-EGFR, anti-MET, anti-HER2). Conclusions: In this series, we observed KRAS wild type gene amp+ to be present in a subset (16%) of GEC patients at diagnosis, correlating with very high protein expression. KRAS amp+ was present after treatment with trastuzumab in HER2+ patients, and also after anti-MET therapy. These data suggest that KRAS amp+ represents a molecular subset with advanced disease at diagnosis. The observation of acquired KRAS amp+ after targeted therapies may be a resistance mechanism to anti-HER and anti-MET inhibitors. Inhibition using combined MEK/AKT pathway inhibitors, and proof-of-principle siRNA, warrants further investigation for KRAS amp+ tumors. Citation Format: Les Henderson, Peng Xu, Brittany Rambo, Wei-Li Liao, Todd Hembrough, Daniel Catenacci. KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A55. doi: 10.1158/1557-3125.RASONC14-A55

Profiling miRNAs in nasopharyngeal carcinoma FFPE tissue by microarray and Next Generation Sequencing

Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous-cell carcinoma that occurs in the epithelial lining of the nasopharynx. Nasopharyngeal carcinoma has a geographically well-defined distribution worldwide, with the highest prevalence in China, Southeast Asia, and Northern Africa. Symptoms of nascent NPC may be unapparent or trivial, with diagnosis based on the histopathology of biopsied tissue following endoscopy of the nasopharynx. The tumor node metastasis (TNM) staging system is the benchmark for the prognosis of NPC and guides treatment strategy. However, there is a consensus that the TNM system is not sufficiently specific for the prognosis of NPC, as it does not reflect the biological heterogeneity of this tumor, making another biomarker for the detection of NPC a priority. We have previously reported on different approaches for microRNA (miRNA) biomarker discovery for Formalin Fixed Paraffin Embedded (FFPE) NPC tissue samples by both a targeted (microarray) and an untargeted (small RNA-Seq) discovery platform. Both miRNA discovery platforms produced similar results, narrowing the miRNA signature to 1–5% of the known mature human miRNAs, with untargeted (small RNA-Seq approach) having the advantage of indicating “unknown” miRNAs associated with NPC. Both miRNA profiles strongly associated with NPC, providing two potential discovery platforms for biomarker signatures for NPC. Herein, we provide a detailed description of the methods that we used to interrogate FFPE samples to discover biomarkers for NPC.

MammaPrint Molecular Diagnostics on Formalin-Fixed, Paraffin-Embedded Tissue

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Comparison of Whole Genome Amplification Methods for Analysis of DNA Extracted from Microdissected Early Breast Lesions in Formalin-Fixed Paraffin-Embedded Tissue

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

BackgroundThe identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.Methodology and Principal FindingsHerein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.SignificanceThis assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.

Assessment of Microrna Expression in Mantle Cell Lymphoma Using High Throughput Techniques

Mantle cell lymphoma (MCL) accounts for 3–10% of all lymphomas and demonstrates a poor clinical response to current therapeutic approaches, with a median survival of 3–5 years. The natural history of MCL is heterogeneous and not well-defined by standard clinical markers as patients may die within months of diagnosis or experience long-term survival. There remains a need for reliable biomarkers of MCL prognosis. To date, global gene expression signatures have not been determined for formalin-fixed, paraffin-embedded (FFPE) MCL samples due to difficulties isolating full-length mRNA transcripts from FFPE tissues. Examining microRNA expression in FFPE samples may circumvent this problem, as this population of RNAs remains intact during processing of FFPE samples. MicroRNAs (miRs) are small, non-coding RNAs, which regulate gene expression by inhibiting mRNA translation. Although miR expression signatures have been derived for other hematological malignancies, assessment of miR expression in patients with MCL has yet to be undertaken. We hypothesize that different pathological subtypes of MCL have unique miR expression signatures, distinct from miR expression profiles of other B-cell non-Hodgkin lymphomas. Our objectives were two-fold:To determine and validate miR expression in different pathological subtypes of MCL; and,To compare miR expression in MCL to other B-cell non-Hodgkin lymphomas.Total RNA was extracted from FFPE samples [17 conventional MCL, 11 blastoid MCL, 4 follicular lymphomas (Grades 1, 2, 3a, and 3b), 1 nodal marginal zone lymphoma, 1 small lymphocytic lymphoma (SLL/CLL) and 3 benign, reactive lymph nodes (normal controls)] using the RecoverAll kit for FFPE tissues (Ambion). RNA was subjected to quantitative real-time PCR (qRT-PCR) for 365 miRs and 3 endogenous control small nucleolar RNAs to obtain miR expression profiles using the TaqMan Low Density Array (TLDA) v1.0 platform (MicroFluidic card, Applied Biosystems). TLDAs were run on the ABI7900 HT analyzer with TLDA upgrade and analysed with RQ Manager software provided by Applied Biosystems. Expression profiles were correlated to pathological subtype, and hierarchical clustering, principal component analysis (PCA), and ANOVA were performed using Partek software [Partek Genomics Suite for Gene Expression Data]. Results indicate that miR expression profiles differ between B-cell, non-Hodgkin lymphoma and MCL samples. Clustering analysis and PCA both demonstrated different profiles between MCL and B-cell, non-Hodgkin lymphomas. Although PCA did not demonstrate significant differences between the conventional and blastoid MCL samples, a set of fifteen miRs may be able to distinguish these two groups of MCL, since these 15 miRs are relatively upregulated in blastoid MCL in comparison to conventional MCL. In addition, PCA revealed five (3 conventional MCL samples and 2 blastoid samples), which did not cluster with their respective groups. These five samples were from patients known to have progressive disease, indicating that such patients may have different miR expression profiles compared to patients with non-progressive disease. We conclude that high-throughput miR expression profiles can be generated from FFPE samples in B-cell non-Hodgkin lymphomas and that miR expression profiles for MCL samples differ from those of other B-cell non-Hodgkin lymphomas. Blastoid and conventional MCL samples may not have significantly differing profiles, however a set of 15 miRs appears to be able to distinguish between these two groups. Of note, samples from patients with known progressive disease have significantly different profiles from those with non-progressive disease.

Sexually transmitted disease control in developing countries: the challenge of involving the private sector.

<h3>Abstract</h3> Treatments of patients with solid tumors frequently rely on the relative and semi-quantitative assessment of tissue protein biomarkers by immunohistochemistry (IHC). The perspective of transition to absolute and quantitative...