Formalin-fixed Paraffin-embedded Cancer Tissue Research Articles

Proteomics analysis of an individual formalin-fixed paraffin-embedded tissue section using isobaric-tag amplification.

The comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) tissues is essential for retrospective clinical studies. However, detecting low-abundance proteins and obtaining proteome-scale data from FFPE samples pose analytical challenges in mass spectrometry-based proteomics. To overcome this challenge, our study focuses on implementing an isobaric labeling approach to improve the detection of low-abundance target proteins in FFPE tissues, thereby enhancing the qualitative and quantitative analysis. We employed an isobaric labeling approach utilizing synthetic peptides or proteins to enable the qualitative and quantitative measurement of target proteins in FFPE tissue samples. To achieve this, we incorporated tandem mass tag (TMT)-labeled recombinant proteins or synthetic peptides into TMT-labeled metastatic breast cancer FFPE tissues. Through this strategy, we successfully detect coexisting CD276 (B7-H3) and CD147 proteins while identifying over 6000 proteins using targeted analysis of individual FFPE tissue sections. Our findings provide compelling evidence that the incorporation of isobaric labeling, along with the inclusion of TMT-labeled peptides or proteins, greatly enhances the detection of target proteins in FFPE tissue samples. By employing this approach, we were able to obtain robust qualitative measurements of CD276 and CD147 proteins, showcasing its effectiveness in identifying more than 6000 proteins in FFPE samples. The integration of an isobaric labeling approach, in conjunction with synthetic peptides or proteins, presents a valuable strategy for enhancing the detection and validation of target proteins in FFPE tissue analysis. This technique holds immense potential in retrospective clinical studies, as it enables comprehensive analysis of low-abundance proteins and facilitating proteome-scale investigations in FFPE samples. By leveraging this methodology, researchers can unlock new insights into disease mechanisms and advance our understanding of complex biological processes.

Abstract A010: ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype?

Abstract Introduction The expression level and single nucleotide polymorphisms of the prostate-specific gene ANO7 have been shown to correlate with prostate cancer (PrCa) outcome. Very little is known about ANO7, apart from its expression profile in tissues and that the protein is either a calcium-activated ion channel or a lipid scramblase. Our results suggest that ANO7 likely has a role in PrCa progression and that loss of ANO7 is associated with malignant PrCa. Materials and methods Sections of a prostate cancer formalin-fixed paraffin-embedded tissue microarray were used for ANO7 mRNA and protein detection. mRNA was detected with fluorescent in situ hybridization (FISH). Protein was detected with immunohistochemistry (IHC) using a polyclonal anti-ANO7 antibody (HPA035730). mRNA from fresh radical prostatectomy primary tumor samples were sequenced and subsequent gene expression levels were analyzed for co-expression with ANO7. The sample set consisted of a matched tumor and benign tissue sample (15+15) from each patient. Results The FISH images indicated explicitly that ANO7 mRNA is exclusively expressed in the luminal cells of the prostatic glandular epithelium. ANO7 mRNA is expressed in all cancerous lumina-forming glandular structures, but not in poorly-formed glands or sheets of cancer, in which ANO7 mRNA expression is very low or non-existent. Morphologically benign glands amidst cancerous tissue showed no expression, in contrast to tissue in which benign glands are predominating. The IHC showed a similar staining pattern to FISH.ANO7 co-expression gene set enrichment analysis resulted in 19 positively correlating and 77 negatively correlating pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) terms. Most of the positively correlating terms relate to metabolism, whereas among the negatively correlating terms, there were many signal transduction and cell growth and death-related pathways, such as Hippo, Ras, and p53 signaling pathways. Conclusions ANO7 mRNA and protein expression correlate spatially in the tissue, showing that the expression is specific to differentiated luminal cells and highly reduced in high-grade cancer. This notion is supported by the co-expressed gene-set enrichment analysis, which is showing a negative correlation of ANO7 with many pathways that are associated with aggressive PrCa. Mainly metabolism-related processes were positively correlated with ANO7, which could indicate that ANO7 is linked to the reprogramming of metabolism in the cells during cancer progression. Further research aims to identify the pathways in which ANO7 plays a significant role and to elucidate how the ANO7 protein functionally alters signaling related to PrCa progression. Citation Format: Olli Metsälä, Gudrun Wahlström, Pekka Taimen, Johanna Schleutker. ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype? [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A010.

In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens.

Protein-protein interactions (PPI) are the basis of various biological phenomena, such as intracellular signal transduction, gene transcription, and metabolism. PPI are also considered to be involved in the pathogenesis and development of various diseases, including cancer. PPI phenomenon and their functions have been elucidated by gene transfection and molecular detection technologies. On the other hand, in histopathological analysis, although immunohistochemical analyses provide information pertaining to protein expression and their localization in pathophysiological tissues, it has been difficult to visualize the PPI of these proteins. An in situ proximity ligation assay (PLA) was developed as a microscopic visualization technique for PPI in formalin-fixed, paraffin-embedded (FFPE) tissues as well as in cultured cells and frozen tissues. PLA using histopathological specimens enables cohort studies of PPI, which can clarify the significance of PPI in pathology. We have previously shown the dimerization pattern of estrogen receptors and significance of HER2-binding proteins using breast cancer FFPE tissues. In this chapter, we describe a methodology for the visualization of PPI using PLA in pathological specimens.

Optimization and Validation of Nanopore Based Sequencing Method for Molecular Testing of CNS Tumours

BackgroundThe World Health Organization (WHO) introduced molecular identifiers for the diagnosis and prognosis of CNS tumors including the mutational status of isocitrate dehydrogenase or IDHgenes in glial tumors. Currently used immunohistochemistry (IHC) is not capable of detecting the non‐canonical mutations, and sequencing is often required as a follow‐up. Current next‐generation sequencing (NGS) technologies used in tumor molecular marker detection introduce key challenges including high capital cost, complex infrastructure requirements, and long turnaround times. These challenges considerably limit the ability to perform NGS testing in many pathology laboratories. In this study, we aimed to use third generation nanopore sequencing technology to resolve these limitations. The Oxford Nanopore MinION, a pocket‐sized nanopore sequencing device, has minimal capital costs and infrastructural requirements, and shorter turnaround times. However, the nanopore technology has not been validated in clinical practice and has not been optimized on formalin‐fixed paraffin‐embedded (FFPE) tissue.MethodsDNA extraction of selective tumor areas was performed from the corresponding FFPE tissue blocks from a cohort of gliomas with confirmed IDH1and IDH2gene statuses (n=65). A PCR amplicon‐based approach was used to amplify hot spots of the IDH1and IDH2genes, starting with 30ng DNA material. The amplicon libraries were sequenced for 2 hours in multiplex on the MinION device and IDHSNPs were called with the Nanopolish software. ASIP Abstract Mashiat MimosaResults26 IDH mutant samples were identified: 21 IDH1R132H, 2 IDH1R132G, 2 IDH2R172G, and 1 IDH2D177H. All cases showed concordant IDHmutational status when compared to the reference methods (IHC or NGS) and both analytical sensitivity and specificity were 100%. Precision analysis of variant allele frequency (VAF) showed the coefficient of variation was less than 5% (both inter and intra runs), and the limit of detection for VAF was 3%. The range of read depth obtained was 882X to 43,000x with an average of 20,000x. This assay revealed a $50‐$100 material cost per sample, and the time taken from extracted nucleic acid to final result generation was 1‐2 business days.ConclusionThis project is the first to optimize and validate an approach to detect SNP mutations in FFPE samples using nanopore technology. It has demonstrated the feasibility and efficacy of the nanopore amplicon sequencing method in cancer FFPE tissue with excellent test performance characteristics, significantly shorter turnaround times at considerably lower costs and without any infrastructural needs. Thus, it can be used to circumvent challenges to current NGS testing platforms and can be the milestone that would make cancer NGS testing available for every laboratory.

Determination of human papillomavirus type in archival tissue specimens of invasive cervical cancer using molecular mapping and E6/E7-based polymerase chain reaction.

In our previous study, an L1-based human papillomavirus (HPV) test using liquid-based cytology revealed that some invasive cervical cancers (ICC) exhibited multiple HPV types or harbored no HPV DNA. Here, molecular mapping of formalin-fixed paraffin-embedded cancer tissue specimens from the same patients were conducted to confirm these observations. Among 377 ICC cases, 73 eligible specimens (9 positive for multiple HPV types, 16 negative for HPV, and 48 positive for a single HPV type from the previous study) were reexamined by manual microdissection of cancer lesions, then subjected to HPV genotyping using the uniplex E6/E7 polymerase-chain-reaction method to detect all high-risk and potentially high-risk HPV types. The HPV typing results were confirmed in 52 of 73 cancer cases; among the 21 remaining cases, 15 were discordant and 6 were partially concordant. In total, 8 of 16 (50%) HPV-negative samples became positive; 6 were positive for HPV16 and 2 were positive for HPV67. Moreover, two samples previously positive for HPV6 and HPV53 were negative for HPV. All nine cancers with multiple HPV types were found to harbor only a single HPV type. In total, 63 cancer tissues exhibited a single HPV type. HPV16 and HPV18 were detected in squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Alpha-5 (HPV82), -6 (HPV56), and -9 (HPV31/52/67) HPV types were detected in SCC, whereas Alpha-7 (HPV59/68) types were detected in ADC and adenosquamous carcinoma (ADSCC). These findings suggested that the different HPV types induced different histological cancers. Furthermore, all SCCs and 10 of 11 usual-type ADCs were positive for high-risk HPV types, supporting the use of HPV screening for the detection of these cancers and associated premalignant lesions. HPV16 is likely to remain undetected in some cervical cancer tissues because of low viral-copy-numbers. Putative high-risk HPV types (e.g., HPV67 and HPV82) might be high risk in Japan.

Novel rapid immunohistochemistry using an alternating current electric field identifies Rac and Cdc42 activation in human colon cancer FFPE tissues

It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine.

Pathogenic variants of homologous recombination repair-related genes in advanced pancreatic cancer and oxaliplatin-based chemotherapy: Prospective multicenter observational study.

555 Background: The latest National Comprehensive Cancer Network Guidelines for pancreatic adenocarcinoma recommended platinum-based chemotherapy for the patients with germline BRCA1/2 or PALB2 variants based on retrospective studies. However, the association between the efficacy of oxaliplatin-based chemotherapy and homologous recombination repair (HRR)-related gene variants has not yet been evaluated in a prospective study. Methods: This was a multicenter, prospective, observational study. Key inclusion criteria were: histologically confirmed pancreatic adenocarcinoma or adenosquamous carcinoma; candidates for systemic chemotherapy or currently under systemic chemotherapy for unresectable disease; age ≥ 20 years; Eastern Cooperative Oncology Group Performance Status 0–2; formalin-fixed paraffin-embedded cancer tissue available for genomic sequencing; and adequate hematological, liver, and renal function. Patients were assessed with the next generation sequencing (NGS)-based ACT-repair panel (ACT genomics; Taipei, Taiwan). ACT-repair panel is accredited by College of American Pathologists and is designed to detect short variants (SVs) including substitutions, insertions, deletions, and copy number variants of 35 genes including 8 HRR-related genes ( ATM, ATR, BRCA1, BRCA2, PALB2, RAD51B, RAD51C, RAD51D). The primary endpoint was the one-year overall survival rate (1yr-OS%) after the initiation of oxaliplatin-based chemotherapy in patients who harbored pathogenic HRR gene variants. On the basis of published retrospective data, expected 1yr-OS% was set at ≥ 60% in this study. Results: Forty patients were enrolled from August 2018 to March 2020. Median age was 67 years (range, 49–81 years). Sequencing data were obtained from 39 patients (NGS success rate = 97.5%). Nine patients (22.5%) harboring HRR gene; ATM SVs (n = 4), BRCA2 loss of heterozygosity (LOH) (n = 3), BRCA2 SVs (n = 1), and PALB2 LOH (n = 1). Three patients received oxaliplatin-based chemotherapy as first-line chemotherapy, while the remaining six patients received it as second- or later-line oxaliplatin-based chemotherapy. The 1yr-OS% was 44.4%, and the median overall survival was 221 days (95% confidence interval, 79–NA days) after the initiation of oxaliplatin-based chemotherapy. In three patients who received oxaliplatin-based chemotherapy as first-line treatment, overall survivals were 703 (alive), 694 (alive), and 405 (dead) days, respectively. Conclusions: Efficacy of oxaliplatin-based chemotherapy on advanced pancreatic cancer harboring HRR-related gene variants did not meet the primary endpoint of 1yr-OS% (≥ 60%). Clinical trial information: UMIN000033655.

Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples.

Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.

RCasFISH: CRISPR/dCas9-Mediated in Situ Imaging of mRNA Transcripts in Fixed Cells and Tissues.

Effective characterization and imaging of endogenous RNA transcripts have important value in the diagnosis, treatment, and prognosis of diseases. Traditional qRT-PCR as a liquid-based RNA detection method might lead to false-negative results due to the admixture of too many nontarget cells. Also, many in situ RNA imaging methods were hindered by long turnaround time and insufficient signals. Here, we describe and evaluate a CRISPR/dCas9-MS2-based RNA fluorescence in situ hybridization assay (RCasFISH) for in situ amplified imaging and quantification of RNA transcripts in fixed cells as well as formalin-fixed, paraffin-embedded (FFPE) tissue sections at a single-molecular level in individual cells. Compared to single molecular FISH (smFISH), RCasFISH yields brighter dot signals and a better signal-to-noise ratio (SNR) with lower costs and less than 1.5 h of hybridization. In addition, by using human epidermal growth factor receptor 2 (HER2) as a model, we quantified individual HER2 mRNA molecules in clinical breast cancer FFPE tissue sections and demonstrated its potential to resolve FISH-equivocal cases. Therefore, RCasFISH may provide a new approach for gene expression studies in basic research and hold the potential for molecular diagnostic applications.

Detection of gene mutations in gastric cancer tissues using a commercial sequencing panel.

Predicting malignancy is important for adequate adjuvant therapy in patients with cancer. Due to cancer being a genetic disease, the detection of gene mutations could be helpful in predicting the prognosis and efficacy of drugs. Gastric cancer is the fifth most common cancer and is the third leading cause of cancer associated mortality worldwide. Mutations in genes may correlate with clinical information in patients with gastric cancer after surgery and, therefore, may be useful for predicting the prognosis of this disease. In the present study, to assess the usefulness of a commercial sequencing panel, TruSeq® Amplicon-Cancer Panel (Illumina), using a next-generation sequencer (Illumina MiSeq), mutation analysis of fresh as well as formalin-fixed paraffin-embedded (FFPE) gastric cancer tissues was performed retrospectively. The study group comprised of 4 patients who underwent gastrectomy for gastric cancer. Cancer and normal stomach tissues were collected immediately following surgical removal. Thereafter, the specimens were fixed in 10% neutral formalin for 24–72 h. Normal and FFPE cancer tissues were histologically examined and confirmed. A total of 3 mutations were identified in the driver genes (KRAS, TP53 and APC) in cancer tissues from 2 of the 4 patients, using fresh samples. In addition, FFPE samples were analysed for the same tissues and the same results were obtained by setting the threshold for the percentage of the mutation rate to avoid detection of pseudo-positive mutations. In conclusion, the sequencing analysis using FFPE-derived DNA samples was successfully performed.

Abstract 753: In situ RNA expression profiling of 1600+ immuno-oncology targets in FFPE tissue using NanoString GeoMx™Digital Spatial Profiler

Abstract Clinical specimens including formalin-fixed, paraffin embedded (FFPE) tumor sections preserve spatial and molecular information of tumor cells and their surrounding microenvironment. This valuable spatial information is loss with bulk RNA-seq, the most prevalent method for gene expression profiling of archival FFPE samples. In contrast, the NanoString GeoMx™ Digital Spatial Profiler (DSP) is a high multiplexing assay that can profile thousands of RNAs from user selectable regions of interest (ROIs) in FFPE sections. Here we introduce a 1600+ gene panel of tumor, stroma, and immune cell-specific content derived from NanoString nCounter® PanCancer series. We compare RNA profiling using our 1600+ immune-oncology panel to the RNA-seq methodology in FFPE samples. DSP RNA in situ probes are photocleavable oligonucleotides tags coupled to hybridization sequences that bind to mRNA transcripts in the FFPE tissue section. We gridded 96 ROIs upon the FFPE section, photocleaved oligonucleotide tags from each ROI were collected and prepared into sequencing libraries with our NGS readout workflow. After sequencing, reads were charted back to each ROI in the tissue section, generating a map of transcript activity within the tissue. We found high concordance between “bulk” DSP RNA (counts from all 96 gridded ROIs) and RNA-seq from the same FFPE block. Individually, however, each ROI showed different expression patterns than bulk and ROI expression patterns clustered based on similar tissue morphology. We further profiled the tumor and microenvironment compartments from multiple FFPE cancer tissues, comparing our 1600+ RNA expression profile to RNA-seq. DSP was able to detect a higher number of genes with low expression within each spatial compartment compared to bulk RNA-seq. These data demonstrate that DSP offers unparalleled sensitivity for large-scale gene expression while preserving critical information about tissue architecture. GeoMx™ DSP technology is for Research Use Only and not for use in diagnostic procedures. Citation Format: Margaret Hoang, Zoey Zhou, Michelle Kriner, Kristina Sorg, Zach Norgaard, Erin Piazza, Chris Merritt, Dae Kim, Joseph Beechem. In situ RNA expression profiling of 1600+ immuno-oncology targets in FFPE tissue using NanoString GeoMx™Digital Spatial Profiler [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 753.

Abstract 4572: Cross-validation between anti-CD3/CD8/FOXP3/pan-Cytokeratin fluorescent multiplex IHC and chromogenic single IHC by digital image analysis for quantifying tumor-infiltrating lymphocytes in colorectal cancer patient samples

Abstract A rising need for the overall goal to decipher cancer development and progression is a better understanding of the dynamics of the tumor microenvironment (TME), which are grounded in the interactions and reciprocal manipulation of cancer, stromal, and immune cells. Tumor-infiltrating immune cells influence the TME, and analyses of immune cell types, densities and locations within the TME using fluorescent multiplex immunohistochemistry (mIHC) and digital image analysis appear promising for establishing prognostic indicators and in helping to identify more personalized anti-cancer therapies, but in order to be applied in the clinical setting the relevant assays require a precise validation. We developed a tyramide signal amplification (TSA)-based fluorescent mIHC assay for the detection of CD3, FOXP3, CD8, and pan-Cytokeratin (pan-CK) in formalin-fixed paraffin-embedded (FFPE) tissue on the Leica BOND RX automated staining platform. Algorithms for digital image analysis of chromogenic and fluorescent mIHC were developed using Visiopharm Oncotopix software, allowing for a distinct quantification of T cell populations within tumor and stroma regions of interest. To ensure the reliability and robustness of the mIHC assay an extensive assay validation was performed on FFPE colorectal cancer (CRC) tissue. Chromogenic IHC of the single markers was analyzed by a pathologist and compared to digital image analysis of chromogenic and mIHC. To ensure a suitable linearity of the assay titration series of the primary antibodies were performed. Furthermore, the repeatability and intermediate precision of the assay were determined. Finally, 50 individual FFPE CRC tissue samples were analyzed by fluorescent anti-CD3/FOXP3/CD8/pan-CK mIHC and digital image analysis. Good correlations were observed between chromogenic and mIHC, as well as between pathologist and digital image analysis, and an adequate specificity, accuracy, linearity, repeatability and intermediate precision of the assay could be demonstrated. Furthermore, the distributions and ratios of the differently labeled tumor-infiltrating T cell populations in 50 CRC tissue samples were in agreement with published literature and allowed for a classification of the samples regarding their immune phenotype. The reliable quantification of immune cell subsets in FFPE cancer tissue samples shown here provides an efficient way of analyzing the lymphocyte composition of the TME at a validation level that is comparable to chromogenic IHC and apparently suitable for an application in the clinical setting. The validated combination of mIHC and digital image analysis may therefore enable a classification of the immune status of CRC patient samples and could help to identify new targets for anti-cancer therapy. Citation Format: Daniel Biljes, Philipp Layer, Nickels Winkler, Nadine Fändrich-Dursun, Hartmut Juhl, Bernd Gromoll, Malik Khenkhar, Philipp C. Uhlig. Cross-validation between anti-CD3/CD8/FOXP3/pan-Cytokeratin fluorescent multiplex IHC and chromogenic single IHC by digital image analysis for quantifying tumor-infiltrating lymphocytes in colorectal cancer patient samples [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 4572.

A user's perspective on GeoMxTM digital spatial profiling.

Characterization of spatial protein expression for multiple targets from a single tissue is difficult to perform, especially due to the limitations of multiplex immunohistochemistry and tissue heterogeneity. Therefore, a new technology is required that permits detailed and simultaneous expression profiling of proteins within a defined region of interest (ROI). To address this unmet need, NanoString Technologies developed a new technology, GeoMxTM digital spatial profiling (DSP), which currently enables simultaneous and guided detection of up to 40 antibodies (probes) from a single formalin-fixed paraffin-embedded (FFPE) tissue. DSP probes are tagged with unique photocleavable DNA oligos that are released after guided ultraviolet exposure in specific ROIs. Digital quantification of the released oligos by NanoString's nCounter® system provides a detailed expression profile of proteins within these discrete ROIs. In this article, we will describe our experience with the GeoMx DSP platform using cancer FFPE tissues. These expression profiles will provide better characterization and understanding of tumor heterogeneity and the tumor micro-environment, enabling the improvement of patient therapy and the identification of potential biomarker signatures. The purpose of this article is to offer potential future users an independent insight into the DSP platform and a comprehensive idea of usability, including advantages and current limitations of the technology based on our current experience with the beta version of NanoString's DSP platform as part of the DSP beta-testing program. The GeoMxTM Digital Spatial Profiling (DSP) platform is a non-destructive technique for regional in-depth protein expression profiling. Using oligonucleotide detection technologies, the GeoMxTM DSP enables simultaneous high-level multiplexing on a single FFPE tissue. Here, we focus on our current experience derived from our biomarker research using the beta version of the DSP instrument.

Recurrence risk evaluation in stage IB gastric cancer with TP53 codon 72 polymorphism.

4044 Background: Post-operative adjuvant chemotherapy is not currently indicated for Stage IB gastric cancer. However, about 10% of these patients experience recurrence and metastasis. Our previous study on a panel of gastric cancer cell lines indicated that TP53 codon 72 polymorphisms may affect the degree of biological malignancy. Hence, we hypothesized that the TP53 codon 72 polymorphisms may have been associated with post-operative survival without adjuvant chemotherapy. In this study, we investigated the risk of recurrence after treatment of Stage IB gastric cancer patients carrying the TP53 codon 72 polymorphism and attempted to identify a subpopulation that should receive post-operative adjuvant chemotherapy. Methods: Among 658 gastric cancer patients who received gastrectomy with curative-intent, 130 Stage IB patients were enrolled in the present study. The TP53 codon 72 polymorphisms of formalin-fixed paraffin-embedded cancer tissue sections were assessed by direct sequencing using originally designed primers. Overall survival rate (OS) and relapse-free survival rate (RFS) were analyzed based on the status of TP53 codon 72 polymorphism "Arg/Arg", "Arg/Pro" and "Pro/Pro". The hazard ratio for each subgroup was compared by TP53 codon 72 polymorphism. All interaction p values were calculated using the likelihood test. Results: Of the 125 patients for whom polymorphism analysis results were available, the 5- and 10-year OS was 84.5% and 63.9%, respectively. The 5- and 10-year RFS was 82.2% and 64.3%, respectively. When the study cohort was divided into two groups according to polymorphism status (i.e., Arg/Arg and Arg/Pro vs. Pro/Pro), both the OS (hazard ratio [HR], 1.968; 95% confidence interval [CI], 0.770-7.430, p = 0.045) and RFS (HR, 1.976; 95% CI, 0.778-7.515, p = 0.033) of the Pro/Pro group across the entire observation period were significantly lower than those for the Arg/Arg and Arg/Pro group. The majority of recurrences in Pro/Pro occurred within three years from the operation. Conclusions: Among Stage IB gastric cancer patients that underwent gastrectomy with curative-intent, post-operative adjuvant chemotherapy may be considered immediately after surgery for patients carrying the TP53 codon 72 Pro/Pro polymorphism.

ΔNp63 to TAp63 expression ratio as a potential molecular marker for cervical cancer prognosis.

p63 is a transcription factor p53 family. Two major isoforms of p63, TAp63 with transactivation (TA) domain and ΔNp63 with truncated TA domain, have been reported to play opposing roles either in tumor suppression or oncogenic function. Little is known about the association of these two isoforms of p63 in the carcinogenesis of cervical cancer. In this study, the mRNA expression levels of TAp63 and ΔNp63 in 40 normal, 30 low-grade squamous intraepithelial lesions (LSIL), 38 high-grade squamous intraepithelial lesions (HSIL), and 52 cervical cancer formalin-fixed paraffin-embedded tissues were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We analyzed the association between the ΔNp63 and ΔN/TAp63 mRNA expression ratio and clinicopathological parameters and compared disease-specific survival of each ΔNp63 mRNA expression and ΔN/TAp63 mRNA expression ratio. The ΔN/TAp63 mRNA expression ratio in cervical cancer showed higher sensitivity than the mRNA expression levels of ΔNp63 (52.0% vs 44.2%). The level of ΔN/TAp63 mRNA expression ratio in precancerous LSIL and HSIL was higher than in normal tissues (P = 0.01 and P = 0.003) and lower than in cervical cancer tissues (P = 0.03 and P = 0.02). Besides, the positive ΔN/TAp63 mRNA expression ratio was associated with bulky tumor size and high expression of Ki-67, the proliferation marker, in cervical cancer (P = 0.04 and P = 0.02). The cervical cancer patients with the positive ΔN/TAp63 mRNA expression ratio showed worse survival compared to those who with the negative expression ratio of ΔN/TAp63 (HR = 5.7, 95% CI: 1.6–19.9). In conclusion, the balance of TAp63 and ΔNp63 is closely related to the carcinogenesis of cervical cancer. The ΔN/TAp63 mRNA expression ratio could be useful as a diagnostic and prognostic marker of cervical cancer.

Increases in Tumor N-Glycan Polylactosamines Associated with Advanced HER2-Positive and Triple-Negative Breast Cancer Tissues.

Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) are evaluated for N-linked glycan distribution in the tumor microenvironment. Tissue sections representing multiple human epidermal growth factor receptor 2 (HER2) receptor-positive and triple-negative breast cancers (TNBC) in both TMA and FFPE slide format are processed for high resolution N-glycan MALDI-IMS. An additional FFPE tissue cohort of primary and metastatic breast tumors from the same donors are also evaluated. The cumulative N-glycan MALDI-IMS analysis of breast cancer FFPE tissues and TMAs indicate the distribution of specific glycan structural classes to stromal, necrotic, and tumor regions. A series of high-mannose, branched and fucosylated glycans are detected predominantly within tumor regions. Additionally, a series of polylactosamine glycans are detected in advanced HER2+, TNBC, and metastatic breast cancer tissues. Comparison of tumor N-glycan species detected in paired primary and metastatic tissues indicate minimal changes between the two conditions. The prevalence of tumor-associated polylactosamine glycans in primary and metastatic breast cancer tissues indicates new mechanistic insights into the development and progression of breast cancers. The presence of these glycans could be targeted for therapeutic strategies and further evaluation as potential prognostic biomarkers.

Abstract 4528: RNAseq RNAaccess is the preferred method for expression profiling of low quality FFPE gastric cancer samples

Abstract Clinical trial tissue samples are a valuable resource for biomarker discovery and are frequently collected as Formalin-Fixed, Paraffin-Embedded (FFPE) blocks. FFPE tissues pose challenges for next generation sequencing (NGS)-based expression analysis due to poor yield from highly fragmented RNA. The objective of the current study was to evaluate and compare RNAseq RNAaccess (RR) and Nanostring (NS) platforms for molecular profiling of highly fragmented gastric cancer FFPE tissue. RR (TruSeq RNAaccess, Illumina) and NS (PanCancer immune) were performed according to manufacturer protocol using RNA extracted from sourced FFPE gastric cancer tissues (N=6) with a range of DV200 scores (6-34%) and case-matched fresh-frozen (FF) tissue (N=6). For NS, 200ng and 400ng RNA input levels were tested. Data quality from the 400ng samples proved to be superior, resulting in two more samples passing QC. A final sample size of 4 matched pairs was utilized for cross-platform comparisons. For RR, 100ng RNA was used. Pearson correlation coefficients were examined for the following comparisons: 1) FFPE vs. FF samples from RR, 2) FFPE from NS vs. FF from RR, and 3) FFPE NS vs. FFPE RR. Analyses were performed on log2 transformed expression data, for all available genes (N= 770 common genes from RR and NS) and an 18-gene IFNγ signature (IFNγ). All correlations were plotted against a range of DV200 scores from the FFPE samples in order to evaluate a potential tissue quality cutoff for RR, specifically at DV200 levels below Illumina guideline of 30. All RR data samples yielded >80% exonic rate in uniquely mapped reads. Conversely, samples with DV200<14 analyzed by NS failed to pass QC due to low fraction of genes detected above background signal. Comparing FFPE to FF tissue from RR, samples with DV200>10 were highly correlated (r≥0.92), globally or for IFNγ. Comparisons of FF RR to FFPE NS were also well correlated (global median r=0.8; IFNγ median r=0.86). Similarly, expression of FFPE RR was well correlated with FFPE NS (global median r=0.81; IFNγ median r=0.89). Altogether, NS and RR data were highly comparable and minimally impacted by DV200 scores below 30. RR generates gene expression profiles from FFPE tissue that are highly concordant with case-matched FF tissues. This study supports using RR for transcriptional profiling of poor quality gastric cancer FFPE samples. Samples below the Illumina suggested DV200 cutoff of 30 generated good quality RR and NS expression data. IFNγ scores derived from NS and RR from the same tissue were highly concordant. However, NS required higher RNA input level to compensate for low DV200 score and no acceptable expression data was generated on samples below DV200 14. This study will be expanded with data on FFPE samples with DV200<10 to identify a lower limit of RNA quality for RR. Citation Format: Emon Elboudwarej, Marianna Zavodovskaya, Xi Zhao, Luting Zhuo, Carrie Baker Brachmann, Jinfeng Liu. RNAseq RNAaccess is the preferred method for expression profiling of low quality FFPE gastric cancer samples [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 4528.

Somatic BRCA1 mutations in clinically sporadic breast cancer with medullary histological features

BackgroundThe role of somatic BRCA1/2 gene mutations in breast cancer is getting increasing attention in view of hereditary disease. The medullary phenotype and triple negative intrinsic subtypes are often, but not exclusively encountered in BRCA1 germline mutated breast cancer, whilst for BRCA2, no association to specific histological features are known. In this study, we addressed the relationship between morphological medullary phenotype and BRCA1/2 somatic mutations in breast cancer without known positive family anamnesis.Methods32 clinically sporadic breast cancers with medullary features were analyzed for somatic BRCA1/2 mutations (all coding exons) with next-generation sequencing technology. Paraffin-embedded formalin-fixed breast cancer samples from all patients were analyzed.ResultsThree of 32 tumors (9%) had pathogenic (ARUP class-5) BRCA1 gene alterations. Two of these pathogenic variants exhibited deletions leading to frameshift mutations (p.Glu23fs, p.Val1234fs), and the remaining single-nucleotid-variant resulted in premature STOP codon (p.Glu60Ter). In one patient, the same pathogenic BRCA1 mutation was detected (p.Glu23fs) in normal breast tissue. Retrospective follow-up in two patients revealed a positive family history for breast cancer and consecutive germline mutation testing confirmed presence of BRCA1 mutations. No somatic pathogenic BRCA2 mutations were detected.ConclusionsBRCA1 mutation testing may be useful in clinically sporadic breast cancer patients with medullary features to identify potential mutation carriers independently from intrinsic molecular subtype. Formalin-fixed paraffin-embedded cancer tissue can undergo testing within a routine molecular-diagnostic setting as a clinical BRCA1/2 mutation screening strategy.

Mass Spectrometry Imaging in Cancer Research: Future Perspectives.

In the last decade mass spectrometry imaging has developed rapidly, in terms of multiple new instrumentation innovations, expansion of target molecules, and areas of application. Mass spectrometry imaging has already had a substantial impact in cancer research, uncovering biomolecular changes associated with disease progression, diagnosis, and prognosis. Many new approaches are incorporating the use of readily available formalin-fixed paraffin-embedded cancer tissues from pathology centers, including tissue blocks, biopsy specimens, and tumor microarrays. It is also increasingly used in drug formulation development as an inexpensive method to determine the distributions of drugs and their metabolites. In this chapter, we offer a perspective in the current and future methodological developments and how these may open up new vistas for cancer research.

Abstract A092: Comprehensive multidimensional analysis of the immune contexture in non-small cell lung cancer

Abstract Tumors form complex ecosystems that include normal epithelial cells, fibroblasts, blood and lymphatic vessels as well as structural components, and importantly, immune cells. The density, location, and organization of various tumor-infiltrating hematopoietic cells of myeloid and lymphoid origin can impact tumor growth, spreading, and clinical outcome of various cancers, including non-small cell lung cancer (NSCLC). Here, we propose to characterize the microenvironment of lung tumors using a unique set of matched fresh tissue single cell suspensions of tumor and normal lung, paraffin-embedded tissues, and peripheral blood plasma and cells obtained at time of surgery in the same patient. We aim to query mechanisms for local immunogenicity and immunosuppression of NSCLC, by defining interactions between tumor cells and various immune cell subsets in the local microenvironment, and by correlating these findings with presence of tumor-associated antigens (TAAs) and their capacity to elicit spontaneous local and systemic immune responses, as well as immune checkpoints known to impair immune responses. We applied multiplexed immunohistochemistry for formalin-fixed paraffin embedded cancer tissues (MICSSS method; n = 40), serological assays for immunogenicity of tumor antigens from plasma samples and supernatants of B cell cultures from the same patients (ELISA; n = 40), as well as mass cytometry on cell suspensions obtained from matched fresh specimens (CYTOF, n = 20) to (1) characterize the composition and organization of the immune microenvironment, (2) identify drivers of immunosuppression in NSCLC and (3) identify the expression profile of TAAs and B cell specificity in NSCLC. We found that TAAs, such as p53, NY-ESO-1, or ERG, were expressed by lung cancer cells in a subset of patients and that protein expression at the tumor site correlated with the antibody (Ab) response found in plasma of matched patients. While circulating plasma Ab titers did not appear to be correlated with the density of infiltrating B cells, potential associations with presence of tertiary-lymphoid structure, plasma cells (CD138+ cytokeratin− cells) or T follicular helper cells (CXCR5+ CD3+ CD8−) are currently being assessed. Association between molecular alterations and immune cell composition and location will also be discussed. We will also quantify expression of checkpoint molecules (PD-L1, VISTA and HHLA-2) on immune and tumor cells. The correlation between checkpoint inhibitor expression and immune cell densities and presence of immune responses will be presented, to assess whether immune cells contribute to the modulation of T cell effector functions. In conclusion, this study is the stepping-stone to a comprehensive understanding of the complex interactions between lung tumors and their immune microenvironment. By using novel multiplexed approaches, we intend to map the immune and antigenic landscape of human NSCLC, to identify mechanisms of early disease immunogenicity and immunosuppression, and to find new prognostic and predictive markers for the development of novel therapeutic strategies and immunotherapy trials in NSCLC. Citation Format: Romain Remark, Yonit Lavin, Adeeb Rahman, Camille Bigenwald, Soma Kobayashi, Christian Becker, Raja Flores, Sacha Gnjatic, Miriam Merad. Comprehensive multidimensional analysis of the immune contexture in non-small cell lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A092.