Formaldehyde-fixed Paraffin-embedded Research Articles

Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology.

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR). The FF90 procedure was validated using flow cytometric analysis and two mAbs recognizing either the native and FFPE-Ag or exclusively the native Ag. C-terminally truncated NGFR (trNGFR)-displaying native and FF90-treated VLPs derived from HIV-1 did not reveal distinctive changes in particle morphology using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Mice were subsequently repetitively immunized with trNGFR-decorated FF90-VLPs and hybridoma technology was used to establish mAb-producing cell clones. In multiple screening rounds, nine cell clones were identified producing mAbs distinctively recognizing epitopes in FF90- and FFPE-NGFR. This POC of a new methodology should foster the future generation of mAbs selectively targeting FFPE-fixed cell-surface Ags.

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 μg/μL) protein preparation for mass spectrometry (MS) based analysis, in which 2 μg/μL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 μg/μL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

Disease-Specific α-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain.

Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.

Multiplex Immunochromogenic Tissue Staining Employing Primary Antibodies from the Same Species.

Chromogenic immunohistochemistry (IHC) serves as an essential assay for the diagnoses of many diseases including cancer. Single-marker IHC detection is the standard used for clinical diagnostic assays. A technology to stain multiple biomarkers chromogenically on a single tissue will also yield contextual biomarker information. Methods to chromogenically stain multiple biomarkers simultaneously employing antibodies from the same species are limited and require complex protocols. Here we describe both manual and automated protocols using the UltraPlex™ mxIHC technology that allows simultaneous detection of up to three biomarkers on a single tissue using a single heat-induced antigen retrieval step in formaldehyde-fixed paraffin-embedded (FFPE) tissue and using primary antibodies from any species.

Identification and validation of prognostic microRNAs involved in breast cancer

Breast cancer is a complex illness with many different types. In the Western world, it is the largest cause of cancer-related death among women. Utilizing quantitative real PCR, the expression levels of nine distinct miRNAs (let-7b, miR18b, miR-21, miR-25, miR-29c, miR-106b, miR375, miR505 and miR-150) were studied (qPCR). To isolate total RNA, researchers used 123 formaldehyde fixed paraffin embedded (FFPE) tissues. TILs (tumor infiltrating lymphocytes) were examined in the samples to see if they had any predictive value. Let-7b’s biological activity in ER negative (MDAMB-231) and positive (MCF-7) breast cancer cell lines was investigated among the miRNAs tested. PNATM inhibitor was delivered for this purpose, and the associated miRNA was evaluated using qPCR. A wound healing experiment was used to investigate the transfected cells’ rate of proliferation and migratory potential. The presence of markers in the let-7b inhibited was examined using immunohistochemistry.

Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.1. Apoptotic associated pathway (FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.2. Epigenetic modifier pathway (GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.3. Translation initiation pathway (DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.4. Transcription factors (MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complexpathogenesis of this rare tumor. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

The Concept of an Epilepsy Brain Bank

Epilepsy comprises more than 40 clinical syndromes affecting millions of patients and families worldwide. To decode the molecular and pathological framework of epilepsy researchers, need reliable human epilepsy and control brain samples. Brain bank organizations collecting and supplying well-documented clinically and pathophysiologically tissue specimens are important for high-quality neurophysiology and neuropharmacology studies for epilepsy and other neurological diseases. New development in molecular mechanism and new treatment methods for neurological disorders have evoked increased demands for human brain tissue. An epilepsy brain bank is a storage source for both the frozen samples as well as the formaldehyde fixed paraffin embedded (FFPE) tissue from epilepsy surgery resections. In 2014, the University of Saskatchewan have started collecting human epilepsy brain tissues for the first time in Canada. This review highlights the necessity and importance of Epilepsy Brain bank that provides unique access for research to valuable source of brain tissue and blood samples from epilepsy patients.

Abstract 179: Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus)

Abstract Comprehensive genomic profiling (CGP) of tumor samples by next-generation sequencing is used to support clinical and translational research into the genetic variants that serve as biomarkers for diagnosis, prognosis and potential therapeutic response. However, as these assays grow in size to meet the expanding demands of users, it is challenging to maintain performance in the face of limited sample input necessitated by small sample volumes and to provide a simple and fast sample to report workflow with limited hands-on time. We, therefore, developed OCA Plus to meet user needs for a large CGP assay with excellent performance. Gene content was prioritized based on potential clinical relevance and variant prevalence in solid tumors. Over 500 genes were selected including genes in the indication statements of approved drug labels, clinical guidelines, and in the enrollment criteria of clinical trials. In addition, driver genes were selected in key pathways including DNA repair and immune checkpoint response. Amplicon design strategies were optimized accordingly for key hotspots, full coding sequences or copy number variation (CNV). The assay used Ion AmpliSeq™ technology with manual library preparation or automated templating on the Ion Chef™ System and sequencing on the Ion GeneStudio™ S5 platform. Twenty ng of purified DNA was routinely used as input. An automated tumor-only workflow for variant calling and sample quality reporting was provided within Ion Reporter™ Software. Streamlined access to reporting of variant relevance was enabled by Oncomine™ Reporter. In development studies of cancer cell line and formaldehyde-fixed, paraffin-embedded (FFPE) tumor samples, the assay displayed excellent uniformity (98% and 94%, respectively). Detection of single nucleotide variants and indels in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Detection of CNV gain and loss in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Assessment of tumor mutational burden (TMB) using publicly available whole-exome cancer sequencing data as well as test cell lines and FFPE samples showed high concordance with whole exome sequencing (R2 &amp;gt; 0.90). MSI sensitivity and specificity was &amp;gt;95% as tested using a diverse set of tumor samples. Targeted fusions were reported with 100% sensitivity and specificity when tested with commercially available controls. Total time from purified DNA to end of sequencing was &amp;lt; 2 days with &amp;lt; 3 hours of hands-on time and the time from post-sequencing to report generation was &amp;lt; 2 hours. Oncomine Comprehensive Assay (OCA Plus) was developed to support CGP and routine clinical research in oncology. The assay design and informatics workflow were optimized to support low input and rapid sample-to-report turn-around time, which will accelerate clinical and translational research. Citation Format: Vinay Mittal, Jennifer Kilzer, Dinesh Cyanam, Janice Au-Young, Santhoshi Bandla, Gary Bee, Sameh El-Difrawy, Aren Ewing, Rajesh Gottimukkala, Mohit Gupta, Nickolay Khazanov, Anelia Kraltcheva, Amir Marcovitz, Scott Myrand, Rose Putler, Yu-Ting Tseng, Warren Tom, Cristina Van Loy, James Veitch, Paul Williams, Elaine Wong-Ho, Huimin Xie, Chenchen Yang, Zheng Zang, Seth Sadis. Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 179.

Abstract 2679: Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues

Abstract Introduction: Imaging mass cytometry (IMC) enables highly multiplexed assessment of more than 30 proteins at a time for the quantification of cell phenotypes within the tumor microenvironment of formaldehyde fixed-paraffin embedded (FFPE) tumor tissue. This methodology provides a deep understanding of the immune-contexture while preserving their spatial information in regards to malignant cells. We report a detailed workflow for optimization of a 27-plex immune-oncology (IO) panel, immune-phenotyping, and spatial analysis of tumor tissue. Methods: IO-panel includes 27 antibodies targeting lymphoid (CD3e, CD4, CD8a, CD19, CD45RO, LAG3, ICOS, Granzyme-B), myeloid (CD11b, CD14, CD33, CD68, CSF1R, IDO-1), immune-regulatory (HLA-DR, OX-40, VISTA, TIM-3, CD73, B7-H3), epithelial (Cytokeratin), proliferative (Ki-67), and constitutive (GAPDH, NaKATPase, Vimentin, aSMA, Histone H3) markers that were selected by singlet automated chromogenic immunohistochemistry (IHC) staining. BSA-free formulation from the same clones of antibodies were isotope-metal conjugated and manual indirect immunofluorescence (IF) staining was used to confirm the stability of metal-tagged antibodies. The optimization by singlet-IF and multiplexed-IMC staining was performed with normal and malignant tissues (4mm core TMA with tonsil, placenta, prostate, breast carcinoma, ovarian carcinoma, and endometrioid carcinoma tissues). All images from IHC (HALO software), indirect IF (InForm software), and IMC (MCD viewer software) slides were evaluated by two pathologists. Cell densities and spatial analysis of a breast carcinoma core was performed using HALO software. Results: All antibodies included in the panel were individually optimized by IHC under the same protocol conditions. A significant correlation of optimal antibody concentrations where observed across the different staining techniques (IHC vs IF, r=0.51; IHC vs IMC, r= 0.54; IF vs IMC, r=0.992; all p values &amp;lt;0.01). The breast carcinoma core (total area=0.308mm2) was segmented into epithelial (area=0.216 mm2) and stroma (area=0.092mm2) compartments by digital image analysis. Individual cell segmentation was obtained with based on iridium nuclei signal (total cells=2420). Automated immune-phenotyping showed 20 different cell phenotypes with a predominance of malignant cells expressing B7-H3 (120.1 cells/mm2) and T-lymphocytes expressing LAG3 (3.2 cells/mm2). The average distance between proliferative cytotoxic T-lymphocytes to malignant cells (MCs) was higher when MCs were proliferating (22.94 μm, and 55.4 μm, respectively). Conclusions: IMC is a powerful platform for highly multiplexed detection of protein and cell phenotypes quantification in FFPE samples. Our developed IMC panel and digital image analysis of tissues allow the quantification of immune cells and spatial analysis of the tumor tissue microenvironment. Citation Format: Pedro Rocha, Luisa Solis, Edwin Parra, Ou Shi, Barbara Mino, Vidya C. Sinha, Amanda Rinkenbaugh, Auriole Tamegnon, Mak Duncan, Wei Lu, Mei Jiang, Lakshimi Kakarala, Jared K. Burks, Helen Piwnica-Worms, Cara Haymacker, Michael Teztlaff, Ignacio Wistuba, Alejandro Francisco-Cruz. Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2679.

Detection of GNAQ/ 11 Mutations in Uveal Melanoma Patients' FFPE DNA with Droplet Digital PCR

To quantitatively detect GNAQ/11 mutations in uveal melanoma (UM) by droplet digital PCR (ddPCR). Formaldehyde-fixed paraffin-embedded (FFPE) tumor samples were taken from 78 UM patients with enucleation in West China Hospital between 2009 and 2015. None of the patients received radiotherapy or chemotherapy before enucleation. A retrospective study was conducted to detect GNAQ/11 mutation in UM by ddPCR. To compare the consistency of the results of the two detection methods, DNA sequencing was performed on the target gene by Sanger sequencing. 78 patients with UM were studied retrospectively. GNAQ/11 mutations in uveal melanoma was detected by ddPCR. The consistency of the results of the two detection methods was analyzed. GNAQ/11 mutations frequency was 91.9%. The consistency test between Sanger sequencing and ddPCR of GNAQ/11 mutations in 74 patients with UM was conducted. Kappa coefficient=0.436, P=0.001. The error rate of Sanger sequencing results was significantly higher in the heterogeneous group than in the homogeneous group (12/37 vs. 3/16, P=0.53), but the difference was not statistically significant. The results of ddPCR and Sanger sequencing showed good consistency, and the mutation ratio of GNAQ/11 in UM was significantly different. GNAQ/11 mutation frequency in UM patients detected by ddPCR was close to the reported frequency. It is more recommended to use ddPCR with high sensitivity to detect gene mutations in samples of tumor tissue DNA derived from FFPE. Sanger sequencing is prone to false negative results.

Identifying the distant metastasis genes by next-generation sequencing panel in nasopharyngeal carcinoma.

e18547 Background: Even though local and regional controls have been substantially improved in nasopharyngeal carcinoma (NPC) in the contemporary era of intensity-modulated radiotherapy with extensive use of combined chemotherapy, the distant metastasis becomes the major cause of treatment failure and cancer-related death. To date, the genes contributed to metastasis of NPC is still unclear. The aim of this study was to identify the genes which lead to distant metastasis. Methods: A total of forty primary nonkeratinizing NPC patients were diagnosed at Shandong Cancer Hospital in this study. The formaldehyde-fixed paraffin embedded (FFPE) taken from primary sites or metastatic lymph nodes were performed next-generation sequencing (NGS) panel (Shanghai OrigiMed Co., Ltd.) to determine variated genes, such as single nucleotide variants (SNV), copy number variants (CNV) and rearrangement. These patients were followed up until Febr. 8, 2020. The genes related to distant metastasis were identified by logistic regression. Moreover, this study compared the frequency of mutated gene between our data and Catalog of Somatic Mutations in Cancer (COSMIC) database by the Chi-square test or Fisher’s exact test. Results: The study included 31 men and 9 women. The median age of the patients at diagnosis was 47 years (range 15–71 years). With the median follow-up of 10.6 months (range 16.8–72.3 months), 7 patients had distant metastasis and 1 undergone recurrent. Notably, EMSY and MCL1 variants were contributed to NPC distant metastasis (OR = 31, P = 0.049). The top eight SNV of genes in our study were CYLD, KMT2D, BAP1, EP300, TP53, ATM, NFKBIA and SPEN. When compared to COSMIC database, the mutant frequencies of CYLD, EP300 and BAP1 in our study were significantly higher than that of COSMIC database. However, the mutant frequencies of IDH2 and KMT2C were significantly lower than COSMIC database. Conclusions: This is the first study which suggests that EMSY and MCL1 variants were involved in the metastasis of NPC. The study identified 5 genes, which mutation frequency is significantly different from the COSMIC database. The study provided a molecular basis for a comprehensive understanding of, and exploring targeted therapies for nasopharyngeal carcinoma.

Abstract 780: High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians

Abstract Background: African-Americans have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive and African-Americans are more than twice as likely to die of the disease compared to Caucasians. To clarify the mechanisms involved in this disparity, we analyzed the role of miR-155 in African-American and Caucasian prostate cancer tissues and cell lines. Materials and Methods: Human tissue samples from African-Americans and Caucasians clinical FFPE (Formaldehyde Fixed Paraffin Embedded) were used to analyzed miR-155 expression by Real Time PCR. We selected prostate cancer cell lines from Caucasians (DU-145, LNCaP, PC3), African-American (E006AA-hT) and normal epithelial cell line (PWR-1E) which express different levels of miR-155, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We knocked down miR-155 in cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For migration analysis, miR-155 knockdown cells were seeded in chambers and we measured the capacity of cell motility towards a chemo-attractant. To identify miR-155 mediated downstream regulators, three target prediction algorithms (miRDB, Targetscan and miRecords) were used. Results: We found miR-155 expression to be higher in African-American prostate cancer tissues compared to Caucasian patients with localized disease. Also, expression of miR-155 in African-American cell line, E006AA-hT, was significantly higher compared to Caucasian cell lines, DU-145, LNCaP and PC3. To determine the role of miR-155 in the differences between African-Americans and Caucasians, miR-155 was knocked down in E006AA-hT and DU-145 cells. miR-155 knockdown in E006AA-hT cells caused a decrease in cell viability compared with negative control, while there was no difference in DU-145 cells. We observed inhibition of migration E006AA-hT miR-155 knockdown cells but not on DU-145 miR-155 knockdown cells, suggesting that miR-155 knockdown have the more potent effect on African-American cell line. We analyzed three target prediction algorithms and found that four genes overlapped, SPI1, JARID2, ETS1 and CEBPB that may play a pivotal role in the treatment of aggressive African-American prostate cancers. Conclusion: We conclude that high levels of miR-155 is associated with prostate cancer aggressiveness in African-Americans. Citation Format: Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Varahram Shahryari, Guoren Deng, Divya Bhagirath, Laura Tabatabai, Sharanjot Saini, Shahana Majid, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 780.

PO-329 Genomic aberration in pancreatic neuroendocrine tumours (PNET)

IntroductionRare tumours are poorly investigated and therefore heavily limited in possible therapeutic approaches, and subsequently therapeutic success. One such tumour entity, representing only 1%–2% of all pancreatic malignancies is Pancreatic Neuroendocrine tumours (PNET). PNET are cancers of endocrine cells of the islets of Langerhans and only a handful of studies thus far have investigated the genetic makeup of these tumours. Apart from surgery and chemotherapy for localised tumours, the only approved targeted drug for treatment of advanced PNETs is Everolimus, specifically targeting the mTOR protein. Much is still lacking in the area of targeted therapies and possible options for treating metastasized tumours. Our aim is to therefore establish a genetic profile of PNET tumours in order to determine more targets connected to mTOR signalling and consequently, increase options for improved therapy approaches.Material and methodsWe performed deep coverage sequencing, averaging a depth of 1000 reads, using an established in-house PNET panel. Our cohort included Formaldehyde Fixed Paraffin Embedded (FFPE) patient samples comprising of 50 primary tumours of varying grades, and 14 metastases.Results and discussionsUsing variant calling pipelines, we determined single nucleotide variations (SNV) and copy number variations (CNV) in our cohort and elucidated potential heterogeneity within the PNET tumours. Out of the 62 sequenced samples, 26 primary tumours, and 6 metastases showed SNV alterations with the most recurring being MEN1 alterations (50%) irrespective of whether the tumour is primary or metastasis, followed by DAXX (27%) which was exclusively mutated in primary tumours. In addition, mutation in a novel candidate, polycomb protein EED has been determined specifically in G3 PNET and the alteration falls in one of the β-transducing repeat of the protein. CNV was determined in 38 samples with a recurring copy number increase for RICTOR (~37%). Intra-tumour heterogeneity, based on allelic frequency, is evident in some samples while the combined SNV and CNV profile of these patients reveal a clear inter-tumour heterogeneity in PNETs.ConclusionOur study reveals previously identified and novel alterations, which require further investigation at the protein level to determine new possible therapeutic approaches for PNET patients. In addition, our data clearly reveals that due to the high level of inter-tumour heterogeneity present in these tumours, it is vital to consider a more personalised targeted approach to therapy.

Abstract 1492: High-efficiency, strand-specific, low-bias library preparation method for transcriptome profiling of low-input RNA

Abstract RNA Sequencing (RNA-seq) is a powerful technique for transcriptome analysis. It is widely used for gene expression analysis, detection of mutations, fusion transcripts, alternative splicing, and the study of post transcriptional modifications. RNA-seq is emerging as a powerful tool for molecular diagnostics as well, providing insights into the biological pathways and molecular mechanisms associated with disease progression including cancer. Recent improvements in next generation sequencing technologies (NGS) and sample barcoding strategies have resulted in significant cost reduction. As RNA-seq is increasingly adopted for molecular diagnostics, the quality and reproducibility of library preparation methods become more important. In addition, demand for library preparation methods that produce successful NGS libraries from very low input RNA or precious clinical samples is increasing. To achieve these goals, we have developed an RNA-seq library preparation method which can be used across a wide range of input RNA (5-1000 ng total RNA). GC content analysis, gene body coverage and gene expression correlation show that these important parameters remain consistent across varying inputs, even though input amounts vary by two orders of magnitude. As a result, our method has increased sensitivity and specificity for low-abundance transcripts, and reduced PCR duplicates and sequence bias, delivering high quality strand-specific data. Our method is compatible with poly A-tail enriched and ribosomal RNA depleted samples as well as RNA extracted from FFPE (formaldehyde fixed paraffin embedded) tissue samples. Citation Format: Anetta Nowosielska-Vecchietti. High-efficiency, strand-specific, low-bias library preparation method for transcriptome profiling of low-input RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1492.

Plasma DNA methylation of p16 and shp1 in patients with B cell non-Hodgkin lymphoma.

Early diagnosis and treatment of non-Hodgkin lymphoma (NHL) are progressively important. It has been shown that aberrant promoter methylation contributes to the development and progression of lymphoma. We tried to explore the effect of methylation of p16 and shp1 genes in plasma in the diagnosis of B-NHL patients. The methylation of p16 and shp1 genes in plasma were detected by methylation specific polymerase chain reaction in 103 patients with B-NHL, and compared with peripheral blood leukocytes (PBLs) and formaldehyde-fixed paraffin-embedded (FFPE) tumor tissues. The results showed that methylation frequency of p16 in plasma, PBLs, and FFPE tumor tissues of newly diagnosed B-NHL patients were 37% (27/73), 16% (12/73) and 39% (16/41), whereas those of shp1 were 47% (34/73), 25% (18/73) and 63% (26/41). High methylation consistency of p16/shp1 between plasma and FFPE tumor tissues were revealed (the values of kappa: 0.84, 0.80). Moreover, there were a higher frequency of methylated p16 in all three samples in patients with B symptoms and lower platelet count (<100×109/L), as well as in patients with stage III/IV in plasma and FFPE tumor tissues. Meanwhile, higher frequency of methylated shp1 was observed in patients with higher LDH level in all three samples. Methylation of p16/shp1 in plasma can represent their methylation status in tumor tissues, and may be promising biomarkers in early diagnosis and prognosis evaluation in B-NHL.

Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.

Introduction and ObjectivesOncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples.Materials and MethodsThe RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.ResultsFGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.ConclusionsWe developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors.

Differential Expression of TCF3 Target Genes Defines Subclasses of Diffuse Large B-Cell Lymphoma with Striking Differences in Clinical Outcome Following R-CHOP Therapy

Background: Diffuse large B-cell lymphoma (DLBCL) is clinically and biologically heterogeneous. Whereas most patients are cured following conventional therapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), a substantial minority are refractory or relapse. Identifying patients at high risk of poor outcomes at the time of diagnosis will make it possible to better choose alternative or experimental therapies. TCF3 (also called E2A) is a transcription factor required in B-lymphopoiesis. Gain-of-function somatic mutations that alter TCF3 or its regulatory partners are associated withBurkitt lymphoma (BL) but not DLBCL. These published findings suggest that biologically-defined, clinically-important subtypes of mature B-cell-derived lymphomas may be distinguished from one another based on differential expression of genes regulated by a master regulator of B-lymphoid biology, namely TCF3. Therefore, in the current study, we hypothesized that biologically- and clinically-important subtypes of DLBCL are distinguishable based upon differential expression of genes that are regulated in B-cells by TCF3.Methods: Fifty-nine subjects who presented with de novo DLBCL at Kingston General Hospital from 2001 to 2010 were identified for inclusion based on the availability of clinical data and sufficient biopsy material for extraction of adequate RNA. We used publically-available microarray-based gene expression and clinical data to identify 99 candidate TCF3 target genes whose differential expression was associated with 3-year overall survival following R-CHOP therapy; these transcripts were represented in aNanoStringCodeSet. Total RNA was purified from cores harvested from representative areas of diagnostic, pre-treatment, formaldehyde-fixed, paraffin-embedded (FFPE) biopsy samples. Twenty-five transcripts whose abundance, individually, was associated with 3-year overall survival in our own cohort were chosen for further study.Results: Unsupervised hierarchical cluster analysis based on relative expression of the 25 TCF3 target genes resolved all except two cases into distinct clusters, denoted A and B, that contained 21 and 36 cases, respectively (heat map at left). In comparing these clusters, Cluster B was associated with rearrangement of MYC (p=0.02) as determined by FISH; no statistically significant associations were evident with age, sex, IPI score, B-symptoms, bulky disease or expression of MYC or BCL2 as determined by immunohistochemistry. Kaplan Meyer (KM) analysis revealed strikingly inferior overall and event-free survival (p=0.0005 for each) for Cluster A cases (KM curve at right).Conclusions: Our findings demonstrate that differential expression of TCF3 target genes ascertained in pre-treatment FFPE biopsy samples of de novo DLBCL defines biologically distinctive subsets of cases with distinctive clinical outcomes following R-CHOP therapy. If validated in an independent cohort, these results may inform both clinical management and the development of new targeted therapies for high-risk cases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1115: Targeted RNA sequencing improves transcript analysis in cancer samples

Abstract RNA sequencing (RNA-seq) is a transcriptome profiling technology that provides multiple levels of insight into the genome. In addition to expression levels (transcript abundance), it generates endpoints such as alternative splicing, somatic mutations and rearrangements, which may have functional consequences in cancer. Although somatic mutations are generally identified by DNA sequencing, RNA-seq has the advantage of detecting allele-specific expression affecting a variant allele, as well as functional chimeric transcripts that result from structural rearrangements. Compared to microarray technologies, RNA-seq can provide additional information about novel transcripts. Due to the complexity of the human transcriptome and the variability of gene abundance, the cost of whole transcriptome sequencing to achieve sufficient coverage to detect these types of alterations remains high. To explore the feasibility of a more cost-effective method, we compared the performance of three different RNA-seq methods: whole-transcriptome-, exome-, and targeted RNA-seq, using RNA derived from cancer cell lines and Formaldehyde Fixed-Paraffin Embedded (FFPE) samples. For whole-transcriptome preparation, we used the Illumina TruSeq Stranded mRNA and total RNA kits for cell line and FFPE samples, respectively. Exome-RNAseq was performed using the Illumina Access kit. The libraries from whole-transcriptome RNAseq were subjected to hybridization capture using OncoPanel-an Agilent SureSelect baitset of 500 cancer-related genes. Compared to whole-transcriptome, exome- and targeted-RNA-seq demonstrated (1) higher coding exon coverage and multiplexing capability; (2) reduced rRNA composition to 1%; (3) comparable gene abundance information and (4) over 90% of reads aligned to coding exon regions in FFPE samples, compare to ∼30% when using whole transcriptome method. In conclusion, we demonstrated that exome- and targeted RNA-seq provide a cost-effective way to analyze a subset of the transcriptome. Furthermore, targeted RNA-seq can be highly multiplexed and is therefore amenable for large-scale tumor profiling in clinical or research settings. Citation Format: Ling Lin, Ryan Abo, Deniz Dolcen, Rachel Paquette, Angelica Laing, Luc de Waal, Aaron Thorner, Matthew Ducar, Liuda Ziaugra, Bruce Wollison, Marc Breneiser, William Hahn, Matthew Meyerson, Paul Van Hummelen, Laura MacConaill. Targeted RNA sequencing improves transcript analysis in cancer samples. [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 1115. doi:10.1158/1538-7445.AM2015-1115

Plasma DNA Methylation of p16 and shp1 in Patients with B-Cell Non-Hodgkin Lymphoma

To discuss the clinical implications of cell free DNA, methylation status of the p16 and shp1 genes in plasma DNA from patients with B-NHL were analyzed. Methylation specific polymerase chain reaction (MSP) was used to detect the methylation status of these genes in plasma, peripheral blood leukocytes (PBLs), and formaldehyde-fixed, paraffin-embedded (FFPE) tumor tissues of 73 B-NHL patients. Results showed methylation frequencies of p16 in plasma, PBLs, and FFPE tumor tissues of B-NHL patients were 37%(27/73), 16% (12/73), 39% (16/41); Whereas those of shp1 were 47% (34/73), 25% (18/73), 63% (26/41). High methylation consistencies of p16/shp1 were observed between plasma and FFPE tumor tissues. In plasma and FFPE tumor tissues, there was a higher frequency of methylated p16 in B-NHL patients who had later disease stage, while no similar association was observed in PBLs samples; Higher frequency of methylated p16 was observed in B-NHL patients who had B symptoms and lower platelet count (<100×109/L) in all three samples. Methylated shp1 was frequently detected in patients with high serum LDH level. In summary, DNA methylation in plasma may be promising biomarkers for B-NHL diagnosis, prognosis evaluation, and targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

A standard protocol for detection of EGFR mutations in cytologic specimens

The aim of this study was to establish a standard protocol for detection of EGFR mutations in cytologic specimens. 287 cytologic samples were collected from the patients who were suspected of having lung cancer at six hospitals in Beijing. A detection protocol for EGFR mutations was designed. Two comparative experiments were carried out for the coincidence in EGFR mutation rates between direct sequencing (Seq) and amplification refractory mutation system (ARMS) methods, and between 40 matched cytologic samples with formaldehyde-fixed paraffin embedded (FFPE) cytologic blocks and cytospin slides. Tumor cells were found in 236 out of 287 cases (82.2%, 236/287) . Among them, there were 31 cases (13.1%, 31/236) of low tumor cell content samples and 205 cases (86.9%, 205/236) of high tumor cell content samples. 180 cases in the high tumor cell content samples (87.8%, 180/205) were diagnosed to be consistent with NSCLC. 25 out of 194 cases were ruled out or indefinite to be diagnosed as NSCLC by immunohistochemistry. By direct sequencing, the mutation rate of EGFR was 27.8% (50/180) in NSCLC samples and 28.2% (50/177) in adenocarcinoma samples (high tumor content samples) . By ARMS, the mutation rate of EGFR was 45.6% (82/180) in NSCLC samples and 46.3% (82/177) in adenocarcinoma samples (high tumor content samples). The EGFR mutation rate in low tumor content samples was 38.7% (12/31) , there was no significant difference in EGFR mutation rates between the groups of low tumor cell content samples and high tumor cell content samples (P = 0.12). The concordance rate of EGFR mutation rates was 100% between scraping tumor cells from slides samples and from FFEP blocks in the 40 matched samples. Forty-eight out of 180 definitive NSCLC patients received Gefitinib therapy. The FPS was 12 months in the gefitinib-treated ARMS⁺ group and 2 months in the ARMS⁻ group (P < 0.001), and the OS was 19 months in the gefitinib-treated ARMS⁺ group and 7 months in the ARMS⁻ group (P = 0.003), but no significant differences were found in the efficacy (PFS and OS) of Gefitinib between Seq⁺ and Seq⁻ groups (P = 0.227, P = 0.510, respectively), and Seq⁺/ARMS⁺ and Seq⁻/ARMS⁺ groups (P = 0.354, P = 0.334, respectively). The detection protocol for EGFR mutations in cytological specimens introduced in this study is tested to be reliable and feasible. Pathological evaluation and immunohistochemistry are important in the detection procedure of EGFR mutations in cytologic specimens. High sensitivity methods should be selected for detection of EGFR mutations in cytologic samples.