For Formalin-fixed Paraffin-embedded Research Articles

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing.

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids' quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6-8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6-8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond.

Abstract 3489: MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs

Abstract Introduction: DNA methylation plays a crucial role in development and progression of cancer. Identification of DNA methylation profiles in cancer is indispensable for early detection. To address this research need, Ion AmpliSeqTM offers targeted next-generation sequencing (NGS) methylation community amplicon panels for DNA methylation profiling in cancer-associated genes. However, the process of designing custom methylation amplicon panels using the Ion AmpliSeq primer design pipeline is intricate, labor-intensive, and time-consuming. Here, we developed a one-step, fully automated, faster, and reproducible nextflow-based primer design pipeline (MetFlow) for development of custom Ion AmpliSeqTM methylation panels. MetFlow streamlines the development of custom methylation panels for cancer-related genes, and thus significantly reduces the time and effort traditionally required for amplicon design. Methods: The fully automated MetFlow pipeline is implemented using Nextflow. MetFlow necessitate an input configuration file for the execution of methylation Ion AmpliSeqTM designs. The configuration file specifies the essential parameters, including target CpG input, reference genome, pool number, and amplicon lengths, which are required for designing the amplicons. The MetFlow pipeline designs candidate amplicons for CpG targets on both Watson and Crick strands, facilitating the subsequent creation of files required for the generation of a methylation panel. Results: The fully automated MetFlow pipeline has been used for designing the customized Ion AmpliSeqTM methylation amplicon panel(s) for cancer and age-related genes. The amplicons lengths in these AmpliSeqTM methylation panels were ranged from 125 bp to 175 bp for formalin-fixed, paraffin-embedded (FFPE) research samples. These customized methylation panels were run on the Ion GeneStudioTM S5 sequencer followed by performance analysis using Ion TorrentTM Suite. The published data for these custom amplicon panels suggest that the Ion AmpliSeqTM has sufficient read depth and mapping efficiency, higher sensitivity and accuracy for methylation measurement, and strong correlation (r=0.993) for methylation profiling among sample replicates. Conclusion: We present MetFlow, an advanced automated primer design pipeline for designing the custom Ion AmpliSeqTM methylation amplicon panels tailored to targeted NGS applications in cancer research. The custom designed Ion AmpliSeqTM methylation panels exhibited sufficient read depth, higher sensitivity and accuracy, and reproducibility. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Renesh Bedre, Drishti Kaul, Anupma Sharma, Andrew Hatch, Loni Pickle, Na Li, Fiona Hyland. MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs [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 3489.

Tumour tissue-derived small extracellular vesicles reflect molecular subtypes of bladder cancer.

mRNA-based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size-exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1g of tissue, incubated with media for 30min. FFPE tumour tissue and tumour tissue-derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV-related bladder cancer-specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26-1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue-derived sEVs may reflect more tissue- or disease-specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy-based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer-specific biomarkers in large cohorts.

Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 μm × 520 μm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼106 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

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.

Fast and reliable Sanger POLE sequencing protocol in FFPE tissues of endometrial cancer.

The new classification of endometrial carcinoma (EC) requires molecular interpretation of somatic polymerase epsilon (POLE) exonuclease domain mutations. The identification of pathogenic mutations within the POLE gene defines the important subtype of ultramutated tumours ("POLE-ultramutated") with specified prognostic and predictive utility. POLE somatic mutations are present in 7-12% of ECs, usually high-grade tumours with aggressive appearance. Molecular analysis of the POLE gene can be performed using a qPCR test, the Sanger sequencing method, a next generation sequencing (NGS) panel test and also in situ hybridisation (IHC) assay. We describe our current approach of identification of POLE mutations using Sanger sequencing technology, which is still the most robust, accurate and fast technique to sequence DNA. We present a reliable protocol for Sanger sequencing of the entire sequence coding exonuclease domain of POLE - exons 9, 10, 11, 12, 13 and 14 (codons 268-491) with 5-10 nucleotides in exon/intron boundaries (reference sequences: NM_006231.4, NP_006222.2). The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues. The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.

Somatic NGS Analysis of DNA Damage Response (DDR) Genes ATM, MRE11A, RAD50, NBN, and ATR in Locally Advanced Rectal Cancer Treated with Neoadjuvant Chemo-Radiotherapy.

Neoadjuvant chemo-radiotherapy (nCRT) represents the standard of care for locally advanced rectal cancer (LARC); however, there exists no biomarker that can predict the cancer's response to treatment as less than 20% of patients experience pathological complete response (pCR). Ionizing radiations induce double strand breaks (DSBs) and trigger a DNA damage response (DDR) involving ATM, ATR, and the MRN complex (MRE11, Rad50, and NBS1). In this study, we performed an extensive mutational analysis of the genes involved in the DDR pathway in LARC patients who have undergone nCRT. 13 LARC patients with pCR and 11 LARC patients with partial response (pPR) were investigated using a NGS dedicated panel, designed for formalin-fixed paraffin-embedded (FFPE) samples, containing ATR, ATM, and MRE11-RAD50-NBN genes. The identified variants were classified according to guidelines' recommendations. Eight non-benign variants, six of which were observed in 3 (23%) out of 13 pCR patients, were identified. In particular, a pCR patient carried out a pathogenetic frameshift mutation in exon 21 of the RAD50 gene. The two remaining non-benign missense variants were found in 2 (18%) out of 11 patients in the pPR group. Our data show that the genes involved in the Homologous Recombination (HR) pathway are rarely mutated in LARC; however, given the identification of a missense mutation in RAD 50 in one case of pCR, it could be worth exploring its potential role as a biomarker in larger series.

Fixed but Feasible: New Opportunities for Molecular Diagnostics in Transplantation.

Fixed but Feasible: New Opportunities for Molecular Diagnostics in Transplantation.

Galactosidase-catalyzed fluorescence amplification method (GAFAM): sensitive fluorescent immunohistochemistry using novel fluorogenic β-galactosidase substrates and its application in multiplex immunostaining.

Multiplex immunohistochemistry/multiplex immunofluorescence (mIHC/mIF) enables the simultaneous detection of multiple markers in a single tissue section by visualizing the markers in different colors. Currently, tyramide signal amplification (TSA) is the most commonly used method because it is heat resistant to multiplexing. SPiDER-βGal (6'-(diethylamino)-4'-(fluoromethyl)spiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3'-yl β-D-galactopyranoside), a novel fluorogenic substrate of β-galactosidase (β-gal) was reported recently. Its properties are favorable for application in sensitive mIF based on quinone methide chemistry. Combining SPiDER-βGal with its related substrates, a novel, sensitive fluorescent IHC method for formalin-fixed paraffin-embedded (FFPE) sections was developed, named the galactosidase-catalyzed fluorescence amplification method (GAFAM). Evaluation of GAFAM indicated the following characteristics: (1) the entire GAFAM procedure was complete within a few hours; (2) the optimal working concentration of the substrates was 20μM; (3) the fluorescent product was heat resistant; (4) the GAFAM exhibited sensitivity comparable with that of TSA, which was higher than that of conventional IF; and (5) the GAFAM was applicable to mIF and multispectral imaging. GAFAM is expected to be applicable to IF (or mIF in combination with TSA), and is a promising tool for facilitating morphological research in various fields of life science.

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 P4-04-06: Integrative analysis of single-cell transcriptomic and spatial profiles characterized distinct tumor microenvironment phenotypes in hormone receptor positive (HR+) breast cancer

Abstract Background Clinical trials have shown minimal activity of immune checkpoint blockade (ICB) in HR+ breast cancer. While this is, in part, attributable to low tumor-infiltrating lymphocyte (TIL) levels in HR+ breast cancer, a thorough investigation of the immunologic aspects of the tumor microenvironment (TME) is required to fully understand the limited efficacy of ICB in HR+ disease. We sought to characterize the TME of HR+ breast cancer at single cell resolution using single-cell RNA-seq (scRNA-seq), paired with tissue-based cyclic immunofluorescence (t-CyCIF), a multiplexed imaging technology for formalin-fixed paraffin-embedded (FFPE) tissue sections across up to 30 distinct antigen channels. We hypothesized that integrative analysis of clinical samples using these technologies would elucidate inter-patient heterogeneity of the TME of HR+ breast cancer. Methods Surgically excised primary tumor samples were obtained from five, treatment-naïve, HR+ breast cancer patients. Each tumor was divided with half preserved as a FFPE block for t-CyCIF and bulk RNA-seq, and half frozen and later dissociated into single cells for scRNA-seq. For each t-CyCIF cycle, slides were stained with 3 antibodies and a DNA dye. The resultant image was captured with a CyteFinder (RareCyte) slide scanning fluorescence microscope. Each slide was stained for 3-7 cycles (9-21 antibodies), depending on tissue stability. Acquired images were processed (illumination correction, alignment, stitching, and segmentation) using mcmicro (https://mcmicro.org/) and the fluorescence intensity per cell per antibody was determined. After identifying regions where cells were appropriately stained throughout the cycles, the tumor, stromal, and immune cell types were determined based on expressed antigens. Samples were further characterized based on the spatial distribution of T cells within the tumor and categorized into three classes: immunologically cold (lacking T cells), immune stromal-restricted (T cells located predominantly in the stromal) and highly inflamed (T cells located throughout entire tumor). CyCIF data was compared to bulk RNA-seq data for each sample. For scRNA-seq, two samples with high cellularity were identified. The single-cell barcoded library was constructed with 10X Chromium and sequenced with NextSeq (Illumina). The cell types were compared as were gene expressions of the corresponding T cell populations between the two samples. For all experiments, downstream analysis was performed in R. Results CyCIF analysis revealed diverse compositions of immune cell types. In particular, the spatial distributions of T cells revealed that the five samples were classified into the three TME classes: 2 immune cold, 1 immune stromal-restricted, and 2 highly inflamed. This trend was consistent in the corresponding bulk RNA-seq data, in which we found higher expression of an interferon-gamma signaling signature in the two highly-inflamed tumors. For the two scRNA-seq samples, one corresponds to the immunologically cold class in CyCIF and the other to the immune stromal-restricted class. Consistent with CyCIF analysis, scRNA-seq revealed that the immune stromal-restricted tumor had higher T cell infiltration than the immune cold tumor, particularly with a higher abundance of naïve T cells, Th17 cells, and helper T cells. Conclusions While HR+ breast cancer is generally considered immunologically cold, our pilot analysis integrating CyCIF, scRNA-seq, and bulk RNA-seq revealed considerable diversity between the five HR+ samples, including the distribution of T cells relative to tumor cells. Additional samples are being collected to allow for more detailed exploration of the TME heterogeneity of HR+ breast cancer. Citation Format: Kenichi Shimada, Yvonne X Cui, Jonathan S Goldberg, Ricardo Pastorello, Janae Davis, Tuulia Vallius, Lukas Kania, Ashka Patel, Mckenna Moore, Esther R Ogayo, Deborah Dillon, Peter K Sorger, Jennifer L Guerriero, Elizabeth A Mittendorf. Integrative analysis of single-cell transcriptomic and spatial profiles characterized distinct tumor microenvironment phenotypes in hormone receptor positive (HR+) breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-06.

Real-Time PCR Assays on Formalin-Fixed, Paraffin-Embedded Medulloblastomas.

Real-time PCR technology has been instrumental in contributing toward biomarker discovery, classification of tumors as well as risk stratification of patients. However, much of its success depends on the quality and quantity of the starting material used for RNA extraction. Clinical samples are most often provided as formalin-fixed and paraffin-embedded, wherein the RNA is extensively degraded, affecting sensitivity. Here, we describe a real-time PCR based assay developed for molecular subgrouping of medulloblastomas that is particularly useful for formalin-fixed, paraffin-embedded (FFPE) samples.

Short report: Performance evaluation of the Idylla\u2122 KRAS and EGFR mutation tests on paraffin-embedded\xa0cytological NSCLC samples

BackgroundQuick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding.Material and methodsThe KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination.ResultsConcordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant.ConclusionOur results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.

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 &amp; 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 &amp; 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.

Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Molecular pathology of tuberculosis : Status, methodology, and limits

In the diagnosis of mycobacterioses, microbiological examination with culture and antibiogram, possibly in combination with molecular biological testing of the fresh material, still represents the gold standard. However, these methods are not available for formalin-fixed paraffin-embedded (FFPE) material or other fixed samples. For this reason, the first step in pathology is to attempt microscopic pathogen detection (ZN/Fite/rhodamine-auramine). Subsequently, molecular pathological examination for the detection of mycobacterial gene sequences should also be considered mandatory today. Although this has clear limits due to the material, it is nevertheless well suited, if carried out correctly, to detect amycobacterial infection or make it unlikely. Anegative result may favor an alternative diagnosis but does not completely rule out mycobacteriosis.For the therapy of tuberculosis or nontuberculous mycobacterial (NTM) disease, the reliable detection of the species and the determination of resistance is of utmost importance. With regard to therapy, the clinician cannot afford to make afalse diagnosis. In case of doubt, arebiopsy for sampling native material, particularly for microbiological testing, should be discussed.

Spatially-resolved proteomics and transcriptomics: An emerging digital spatial profiling approach for tumor microenvironment

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.

Imaging Mass Cytometry Reveals the Spatial Architecture of Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemias

Histologic review of bone marrow trephine biopsies is a central component of the diagnostic and treatment response evaluation of hematologic malignancies. Well-validated antibody reagents are routinely used for immunohistochemistry of these samples to provide additional insight into abnormal antigen expression. However, current immunohistochemistry staining protocols are typically limited to only one or two markers simultaneously. Dysplastic changes in cellular morphology and dyssynchronous expression of lineage markers are common features of myelodysplastic syndromes, myeloproliferative neoplasms and secondary acute leukemias. We have integrated the use of multiple diagnostic validated antibody clones with additional antibodies for hematologic lineages and structural proteins to create a 30-marker panel for imaging mass cytometry (IMC). Antibodies included in this panel identify myeloid, lymphoid, erythroid, macrophage, vascular, megakaryocyte and stromal markers as well as markers of cellular proliferation and apoptosis. Through conjugation to elemental metal tags, the entire panel is stained simultaneously on the tissue sample, then acquired by time-of-flight mass spectrometry on a Hyperion instrument (Fluidigm). Antibody staining concentrations and antigen retrieval conditions were optimized for formalin-fixed paraffin-embedded (FFPE) bone marrow to obtain consistent staining for all markers on the panel. Redundant markers for cell populations were selected to provide further internal validation of the observed staining patterns. After data acquisition, cell segmentation algorithms using CellProfiler and ilastik were applied to quantify marker expression in single cells and Phenograph in HistoCAT was used for cell population clustering. Cluster identities for all cells are associated with the original image location in order to plot the spatial arrangement of populations. Using this highly multiplexed panel, we have imaged sets of bone marrow specimens from patients with normal bone marrow morphology and those with myeloid malignancies. We initially confirmed the staining patterns expected for each antibody patterns of co-expression of lineage markers in normal bone marrow samples. We then extended this panel to examine biopsies from patients with myelodysplastic syndrome, myelofibrosis, and secondary acute myeloid leukemia. We found a clear population of CD71+ CD235a+ erythroid cells with strong expression of the proliferative marker Ki67 located within erythroid islands in normal bone marrow samples and MDS. Cell markers of apoptosis and DNA damage are scattered at low frequency throughout the bone marrow in samples with normal bone marrow morphology, but increased clusters of the DNA damage marker phospho-H2AX are observed in selected cases of myelodysplastic syndromes. Overall, this IMC imaging approach is able to extend the current clinical immunostaining for myeloid malignancies by identifying all major bone marrow cell populations. Through highly multiplexed analysis of bone marrow cell populations, the spatial architecture of cell populations and stromal structures can be elucidated, including erythroid islands, lymphoid aggregates and changes in vascular structures with increasing severity of myelofibrosis. In ongoing studies, the development of these imaging techniques for analysis of archived FFPE bone marrow samples is being applied to translational research on hematologic diseases. Disclosures Oh: Kartos Therapeutics: Consultancy; Disc Medicine: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Blueprint Medicines: Consultancy; Novartis: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy.

Abstract 6186: Protein panel for predicting the efficacy of neoadjuvant therapy for rectal cancer based on proteomics

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and more than 1/3 of all cases are rectal cancer.With the development of early diagnosis and precise treatment, the 5-year survival rate of rectal cancer has been greatly improved. Neoadjuvant radiotherapy and chemotherapy, as the standard treatment for locally advanced rectal cancer, have been included in the NCCN guidelines. However, due to tumor heterogeneity,the response rate to neoadjuvant radiotherapy and chemotherapy varies, and there is no effective method to predict the efficacy of neoadjuvant therapy for rectal cancer. The recent advance of pressure cycling technology (PCT) and data-independent acquisition mass spectrometry (DIA-MS) allows high-throughput and reproducible proteomic quantification for formalin-fixed paraffin-embedded (FFPE) biopsy-level tissue proteome. Methods: We collected 33 patients with rectal cancer who were treated with long-term radiotherapy and capecitabine chemotherapy in our center as the training cohort. Among them, a total of 23 cases with both pre-treatment endoscopic biopsy specimens and surgical pathological sections available were procured and reassessed the Tumor Regression Grade (TRG) after treatment as mentioned above. FFPE tissue samples from each individual were collected with two biological replicates. We further collected other 31 patients from another two independent hospitals as the validation cohort. All the samples were processed by PCT-DIA for proteomic profiling. Candidate proteins were selected from training cohort and confirmed in the validation cohort, as well as Immunohistochemistry (IHC). Results: A total of 5055 SwissProt proteins are quantified with high confidence and a high degree of reproducibility (R2=0.983) from 46 samples in the training cohort. Patients in training cohort were divided into two groups according to responsiveness to radiotherapy (TRG1+2: no significant benefit from radiotherapy; TRG3+4: significant benefit from radiotherapy) to explore differentially expressed proteins (adjusted p value &amp;lt; 0.05, fold change &amp;gt; 2). 35 up-regulated proteins and 114 down-regulated proteins were detected in the responded group. The suppressed proteins were mainly involved in immune response and cell activation, while the elevated proteins mostly participate metabolic processes. Six differentially expressed proteins were further confirmed in the validation cohort using DIA-MS and IHC. More details will be presented. Conclusion: We developed a predictive model composed of 6 proteins which exhibited promising roles for the responsiveness to neoadjuvant therapy in rectal patients. Citation Format: Kailun Xu, Biting Zhou, Xi Zheng, Yingkuan Shao, Jian Wang, Tiannan Guo, Shu Zheng. Protein panel for predicting the efficacy of neoadjuvant therapy for rectal cancer based on proteomics [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 6186.

Abstract C031: NanoString nCounter-based gene expression assay for evaluation of breast cancer molecular subtypes in Ethiopian patients

Abstract Introduction: The burden of breast cancer is escalating across the globe and becoming a major public health problem in Ethiopia. Considering the fact that breast cancer is a heterogeneous disease in its nature, it demands a coordinated multimeric approach for diagnosis, treatment and management. Therefore, NanoString nCounter-based gene expression analysis might be employed for breast cancer intrinsic molecular subtyping, since it is a robust, highly reproducible tool and allows to simultaneously explore the expression of hundreds of genes in a single reaction tube. The aim of the study was to determine the intrinsic molecular subtypes of breast cancer samples from Ethiopian patients using a tailored NanoString nCounter gene expression assay for formalin-fixed, paraffin-embedded (FFPE) material. Methods: The present study was carried out to determine the molecular subtypes of breast cancer in Ethiopian patients. Archived FFPE tumor samples from public hospitals in Ethiopia were retrieved and blocks with clinical and pathologic data were used. From each sample total RNA was extracted by miRNeasy kit, then total RNA concentration and quality were determined and high-quality RNA was subjected to NanoString-based nCounter gene expression analysis using a tailored assay consisting of molecular and immunologic markers. For each gene, gene-fold change expression is given when compared to the reference cut-off value. The study focused on the analyses of estrogen receptor 1(ESR1), progestrone receptor (PGR), erythroblastic oncogene B (ERBB2) genes expression and the intrinsic molecular subtypes done using PAM 50 assay. Statistical analysis was carried out using SPSS software. Results: A total of 164 FFPE breast cancer blocks were analyzed for protein (ER, PR and Her2) expression status and mRNA expression levels. 86/164 (52.4%) breast cancer patients were tumor grade 3. More than 50% of tumor samples were ER and PR positive. In contrast to hormone receptors, only 20 (12.2%) breast tumor samples were found to be positive for HER-2. With regard to the mRNA expression levels of the respective genes, 106 (64.6%), 43 (26.2%) and 143 (87.2%) of breast cancer lesions analyzed showed an upregulation of ESR-1, PGR and ERBB2 genes, respectively. The samples with ER and PR positive results demonstrated by immunohistochemistry (IHC) showed nCounter levels from -60 to 124 (median=7) and -379 to 9 (median= -2), respectively. Samples with HER-2 positive results by IHC showed nCounter levels from -1 to 60 (median=17.5). Using PAM50 algorithm, we found 32 (19.5%) luminal A, 33 (20.1%) luminal B, 24 (14.6%) HER-2 enriched, 29 (17.7%) basal and 17 (10.4%) normal type breast cancer. Conclusion: According to the RNA expression assay, the majority of breast cancer subtypes were luminal A and luminal B and the lowest was normal type. In addition, the large majority of the cases analyzed were hormone receptor positive. Thus, hormonal therapy is of high importance for breast cancer care in Ethiopia. Citation Format: Zelalem Desalegn Woldesonbet, Martina Vetter, Meron Yohannes Nigussie, Tamrat Abebe Zeleke, Yonas Bekuretsion, Mahlet Arayselassie, Mathewos Assefa, Abebe Bekele, Endale Anberber, Claudia Wickenhauser, Eva J. Kantelhardt, Jürgen Bukur, Barbara Seliger. NanoString nCounter-based gene expression assay for evaluation of breast cancer molecular subtypes in Ethiopian patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C031.