Abstract LB-288: Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

Spatial genomics and digital pathology continue to advance at a rapid pace, generating large amounts of data. There is a critical unmet need for advanced tools to expedite spatial data generation, visualization, and analysis, to facilitate an improved understanding of health and disease, especially with the advancement of personalized cancer treatments. In this study we present a novel method for the extraction and analysis of genomic information from specific regions of interest (ROI) within a series of formalin-fixed paraffin embedded (FFPE) breast cancer adenocarcinoma tissue sections. An artificial intelligence-based tumor profiling algorithm first identified tumor, stroma, inflammatory, and necrotic regions within the tissue, and segmentation results were confirmed by a Board Certified Pathologist. Automated lysate extraction was then performed in the selected ROIs using ink-jet technology and novel ink chemistry. The process was performed directly on the tissue slide and the crude lysates generated were transferred to a standard collection tube for subsequent sequencing. DNA was extracted from the selected areas before being purified and analyzed using a multi-cancer panel. Library QC showed optimal coverage at an average of >95% uniformity. Sequencing results showed an average coverage of 95% at 250x across all amplicons of the panel. Variant calling was successfully performed. Sequencing data is presented to demonstrate the high coverage achieved using this workflow as well as the resulting tumor heterogeneity. This novel technology provides an efficient solution for the enrichment of tumor content with high sequencing coverage from FFPE tissue sections. Novel spatial data visualization and analysis techniques allowed a comprehensive assessment of tumor heterogeneity. Further studies on both DNA and RNA should be conducted to validate this approach for clinical molecular testing. Citation Format: Bidhan Chaudhuri, Katie Konigsfeld, Marisa Sanchez, Jason Smith, Casey Laris, Claire Weston, John Butler. A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity [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 6575.

We have recently developed a MALDI imaging mass spectrometry (MALDI-IMS) method to spatially profile N-linked glycans in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs). Tissues are incubated with peptide N-glycosidase, and released N-glycans are detected directly using MALDI-FTICR, linked directly with tissue histopathology. Other methods to detect the localization of glycans in tissues rely on detection of broader glycan structural motifs (i.e., lectins or carbohydrate antigen antibodies), whereas our method is able to simultaneously identify and distinguish 40 or more components of the N-glycome on a single slide. To demonstrate the ability of MALDI-IMS to generate biomarker panels, pancreatic cancer tissue blocks and six TMAs containing matched tumor and non-tumor regions from over 70 patients were profiled. Aberrant glycosylation, such as elevated CA-19-9, is well documented in pancreatic cancer, making this an ideal sample set. To best analyze this complex data set, we developed an in-house analysis script that can extract spectra from tissue cores, and return non-biased statistics for observed glycan ions. Panels were generated using a training set of data and tested on external validation data set. The most accurate panel of 12 glycans achieved an overall sensitivity of 92.9% and specificity of 86.7%. Structural identification of N-glycans has been confirmed by techniques such as on-tissue CID, ethylation analysis, sequential glycosidase digestions, and comparison to glycan structural databases. Furthermore, imaging of entire FFPE sections matched with histological analysis revealed a vast diversity of N-glycan localizations, which could distinguish not only tumor from normal tissue, but also regions of pancreatitis and pancreatic intraepithelial neoplasia. The generated glycan tissue maps will also be used to determine specific glycoprotein carriers of biomarker candidate glycans in tissue and biofluid samples. Citation Format: Richard R. Drake, Thomas W. Powers, Benjamin A. Neely. A MALDI imaging mass spectrometry approach using tissue microarrays to identify an N-glycan biomarker panel for pancreatic cancers. [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 540. doi:10.1158/1538-7445.AM2015-540

Introduction: PAXgene Tissue (PAXgene) is a new, formalin-free fixation technology developed for simultaneous preservation of morphology and biomolecules in tissue samples. PAXgene fixed, paraffin embedded (PFPE) tissue is suitable for conventional histochemical and immunohistochemical staining as well as for extraction of high quality nucleic acids. Material and Methods: Cases of human lung, breast and colorectal cancer were divided and fixed in formalin, PAXgene, or snap frozen in liquid nitrogen (LN2). PAXgene fixed, stabilized specimens were stored for up to four days at room temperature prior to processing. Formalin fixed, paraffin embedded (FFPE) and PFPE tissue morphologies were compared using H&E stain. RNA, miRNA and DNA were isolated from FFPE, PFPE and LN2 tissue. RNA expression was analyzed in 96-well RT-qPCR arrays with pre-designed assays, and miRNA expression profiling was performed with single Sybr-green or primer/probe based assays. DNA was analyzed by agarose gel electrophoresis, long-range, and multiplex PCR. Results: Morphology of PFPE samples was similar to or indistinguishable from FFPE tissue. A high correlation of both RNA and miRNA gene expression results was observed between PFPE and LN2 samples. For all PCR assays, Ct values were within +/− 2 Ct. In contrast, poor correlation was observed between RNA from FFPE compared to RNA from LN2 with differences of up to 10 Cts. Correlation of FFPE to LN2 was higher in the case of miRNA but was still not as high as that of miRNA from PFPE. High molecular weight DNA which performed well in PCR could be isolated from LN2 and PFPE tissue, but DNA from FFPE failed in long-range PCR and showed biased or no amplification in multiplexed PCR. Conclusion: The PAXgene Tissue fixation technology preserves morphology and nucleic acids in tissue samples. It enables morphological and multimodal research biomarker testing with one sample. PAXgene Tissue is for research use only. It is not intended for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4071. doi:1538-7445.AM2012-4071

Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. We have developed methods for small RNA and whole transcriptome analysis of FFPE samples on the Ion Torrent PGM™ System using Ion total RNA-Seq Kit v2 and RiboMinus Eukaryote System v2. Paired FFPE and fresh-frozen RNA samples from lung adenocarcinoma tissue were used in this study. Small RNA libraries were prepared using the Ion Total RNA-Seq kit v2 with modified protocol, followed by sequencing on PGM™ system. Results show that small RNA extracted from FFPE sample was successfully converted to small RNA library. Although percentage of reads mapped to miRBase was slightly lower when compared to fresh-frozen sample, the overall miRBase coverage and detection sensitivity were comparable. For whole transcriptome analysis, RiboMinus Eukaryote System v2 kit was used to deplete rRNA from FFPE samples, followed by library construction using the Ion Total RNA-Seq kit v2 with a modified protocol. Significant reduction in rRNA reads was observed after sequencing. High RefSeq correlations were observed between fresh-frozen and FFPE samples. In conclusion, a full spectrum of gene expression data, from both small RNA and whole transcriptome, were generated from FFPE samples using Ion Total RNA-Seq kit v2 with modified protocols. High correlations were observed between paired fresh-frozen and FFPE samples, demonstrating the reliability of the modified workflow for gene expression profiling on FFPE samples. This study provides feasibility for systematic gene expression analysis on FFPE samples from cancer and other diseases. Citation Format: Jian Gu. Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2013-4127

DNA and RNA have been used as markers of tissue quality and integrity in tissues the last decade. In this research study, kidney, liver, colon, lung, and breast genomic quality were analyzed in tissues from surgical cancer patients and post-mortem cases obtained with an IRB approved protocol and informed/or next of kin consent. DNA extraction was performed on formalin-fixed paraffin-embedded tissues (FFPE) from both groups of cases from the 1970's through the 2000's and formalin fixed / long term 70% ethanol storage(15 + years). To extract the DNA from the FFPE tissues, we soaked all tissues in xylene to remove the paraffin, digested the tissue with a proteinase K solution, and used an automated extraction machine to extract the DNA. A similar SOP was used for the formalin fixed/ 70% long term ethanol-stored tissue, except the tissue was first homogenized rather than soaked in xylene. DNA quality and yield were evaluated using a spectrophotometer. Gel electrophoresis was used to evaluate the length of the DNA in about 20% of the 200 samples. Samples that had optical density (OD) readings above 1.60 and below 2.10 with a DNA yield above 100 µg/mL were deemed to be high quality DNA. FFPE tissues yielded the highest DNA concentration and quality (OD reading of 1.87 and yield of 625 ug/ml) with greater than 95% of the samples meeting the above specifications, while the formalin fixed/long term 70% ethanol stored tissue yielded both the lowest DNA concentration and quality (yield of 52 ug/ml and OD reading of 1.48). In conclusion, the results show that high quality DNA can be extracted from FFPE tissue using an automated DNA extraction procedure with paraffin blocks from the early 1970's to present, and the DNA extracted is useful for clinical and experimental studies. The DNA from tissues stored in aqueous media (formalin fixed / long term 70% ethanol storage) may be useful in some research applications, but the DNA is partially degraded compared to the tissue stored in paraffin blocks. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3203. doi:1538-7445.AM2012-3203

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. Nevertheless, it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissue of interest are limited. Here, we present F FPE tissue with a ntibody-guided c hromatin t agmentation with sequencing (FACT-seq), highly sensitive method to efficiently profile histone modifications in FFPE tissue by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We showed a very small piece of FFPE tissue section containing ~4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. In archived FFPE human colorectal and human glioblastoma cancer tissue, H3K27ac FACT-seq revealed disease specific super enhancers. In summary, FACT-seq allows researchers to decode histone modifications like H3K27ac and H3K27me3 in archival FFPE tissues with high sensitivity, thus allowing us to understand epigenetic regulation. Graphical abstract: ( i ) FFPE tissue section; ( ii ) Isolated nuclei; ( iii ) Primary antibody, secondary antibody and T7-pA-Tn5 bind to targets; ( iv ) DNA purification; ( v ) In vitro transcription and sequencing library preparation; ( vi ) Sequencing.

Microplastic (MP) pollution is increasingly acknowledged as a critical environmental and public health issue. This study sought to establish a robust, clinically compatible method for detecting MP particles in deparaffinized formalin-fixed paraffin-embedded (FFPE) human colon tissue sections, using protocols compatible with routine clinical pathology. We employed mid-infrared photothermal (MIP) microscopy—also referred to as optical photothermal infrared (OPTIR) spectroscopy—as a non-destructive, high-resolution technique for chemical characterization and spatial mapping of polymer particles in intact FFPE samples. Following OPTIR analysis, identical sections underwent hematoxylin and eosin (H&E) staining to facilitate precise histopathological evaluation in defined regions of interest. Using this integrated workflow, we detected and localized polyethylene (PE), polystyrene (PS), and polyethylene terephthalate (PET) particles (21 PE particles, 1 PS particle, and 1 PET fiber) within distinct tissue areas. Subsequent histological assessment revealed characteristic inflammatory features near to these identified MP particles. To our knowledge, this represents the first demonstration of a diagnostic workflow that enables combined infrared spectroscopic and histopathological analysis of MPs in routinely processed human FFPE tissue. This approach offers a promising avenue to elucidate the role of microplastic accumulation in human disease and supports further investigation into potential mechanistic links between MP exposure and inflammatory processes in the colon.

e21135 Background: To understand the dynamics of anti tumor immune responses (IR) in the setting of the ageing immune system, we examined immunologically relevant gene transcripts in formalin fixed paraffin embedded (FFPE) primary cutaneous melanoma (PCM) samples derived from five young (age <50 years) and five elderly (age >or=70 years) patients by qRT-PCR. Methods: A total of 10 FFPE tumor blocks were collected between 2004-2007 of patients with PCM at the University of Connecticut Health Center. Total RNA was extracted from FFPE tissue sections and random primed cDNA was generated. Pre amplification using pooled Taqman assays and Universal PCR Master Mix (Applied Biosystems, USA) was performed for 14 cycles to ensure an adequate amount of starting material for qPCR. Using 15 ng total RNA, the quantification of human IR genes (IL-2, INFg, TNFa, TGFb1, IL-10, Foxp3, and IDO-1) and transcripts of succinate dehydrogenase (SDHA) (as endogenous control) was accomplished by qRT-PCR amplification of a cDNA using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes. All reaction assays including that of SDHA were performed in triplicate on an ABI 7500 Fast system (Applied Biosystems). Cycle threshold (Ct) values for each mRNA were normalized to SDHA (DCT) and represented as RQ=2-DCT. The average RQ was calculated for all ten samples in both cohorts and Student’s T-test was performed to evaluate the statistical significance of the IR gene expression. Results: While no significant difference of expression of Th1 cytokines (IL-2, IFNg, TNFa) or Th2/3 cytokines (IL-10, TGFb) could be detected within the two groups, FOXp3 expression was significantly decreased in PCM in the elderly. Conclusions: Our results suggest ageing may be associated with less regulatory responses in situ in PCM and that more expanded analyses of the nature of the IR in PCM are likely to provide information with implications on prognosis and immune based treatment options for elderly PCM subjects.

Background: Murine double minutes-2 (MDM2) protein plays a key role in physiological processes like growth arrest, senescence and apoptosis. MDM2 negatively regulates key proteins like p53 by inhibiting its transcriptional activity and promoting its proteosomal degradation through ubiquitinylation. In turn, the MDM2 expression is regulated in part by a p53-responsive promotor. This negative feedback control mechanism assures that both p53 and MDM2 are kept at very low levels in proliferating cells. However, in about half of all human tumors, the normal regulation of p53 might be disrupted through direct overexpression of MDM2 caused by e.g. MDM2 gene amplification or a T to G substitution (SNP309) in the promotor region of MDM2. High resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA mutations. The current study aimed at validating and implementing HRMA for screening of colorectal cancer patients (CRC) to detect MDM2 promotor SNP309. Pyrosequencing was used to confirm and characterize HRMA results. Materials and Methods: First, HRMA sensitivity was established using a cell line and a FFPE (formalin-fixed paraffin embedded) dilution model. Then, the sensitivity of pyrosequencing to detect MDM2 promotor SNP309 was evaluated using the same dilution models. Next, HRMA was validated on 10 cell lines. Since the MDM2 SNP309 status for these cell lines was unavailable, all HRMA results were confirmed and characterized by pyrosequencing. Results: The cell line dilution model revealed a detection limit of 6% while the detection limit in a background of FFPE wild-type DNA was found to be 3%. The detection limit of pyrosequencing to analyze SNP309 in a background of wild-type cell line DNA seems to be between 20% and 15%. In a background of FFPE wild-type DNA, the detection limit seems to be 3%. HRMA revealed abnormal melting patterns in 5/10 cell lines and pyrosequencing confirmed the presence of 2 homozygous (G/G) and 3 heterozygous (G/T) genotypes. Preliminary results on FFPE material from CRC patient samples showed abnormal melting patterns in 6/13 samples. These are currently being confirmed using pyrosequencing. Discussion: HRMA was found to be a fast, efficient, sensitive and reproducible screening method for MDM2 promotor SNP309 detection with a detection limit between 3% and 6%, which is higher than that of conventional sequencing methods. Preliminary results indicate that HRMA can be used as a screening method for MDM2 SNP309 by which DNA from FFPE tissues can be tested. Currently, a cohort of CRC patients is being tested for the presence of SNP309 using HRMA and pyrosequencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2103. doi:1538-7445.AM2012-2103

ABSTRACTA variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek’s disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek’s disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek’s disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Background: Black women with residual estrogen receptor (ER)-positive breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence free survival (DRFS) compared to White women. Distant metastases result from motile breast cancer stem cells (BCSCs) disseminating through intravasation portals on blood vessels called tumor microenvironment of metastasis (TMEM) doorways. Each TMEM doorway is composed of an actin-regulatory protein (Mena) expressing cancer cell, a macrophage expressing high levels of receptor tyrosine kinase TIE2, and an angiopoietin-expressing endothelial cell. High TMEM doorway density predicts metastatic potential in patients with ER+/HER2- breast cancer. The function of TMEM doorways can be pharmacologically blocked using TIE2 inhibitors. We hypothesized that racial disparity in DRFS in patients with residual ER-positive disease post-NAC is due to difference in density of TMEM doorways, BCSCs, and motile cells in the residual breast tumor microenvironment (TME). Methods: We performed a retrospective, multi-institutional study of the TME in the residual disease post-NAC from 200 patients. Triple immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tissue sections was used to visualize TMEM doorways. Immunofluorescent staining of FFPE tissue sections for SOX9 and MenaINV was used to visualize BCSCs and motile cells, respectively. The density of TMEM doorways, BCSC and motile cells was determined by automated image analysis. The relationship between the density of TMEM doorways, stem and motile cells, and DRFS was examined using Kaplan Meier log-rank test. Comparisons were made for the entire cohort, and separately for the two most prevalent molecular subtypes: ER+/HER2- and triple negative (TN). The analysis included 200 samples of residual breast cancer post-NAC: 100 from Black and 100 from White women. Results: Black compared to White women were more likely to develop distant recurrence (49% vs 34%, p=0.04), to receive mastectomy (70% vs 51%, p=0.009), and to have TN subtype (35% vs 21%, p<0.0001). Median time-to-follow-up was 70 months (range 2.8-160.7), and was similar between Black and White patients (67.7 vs 71.0 months, p=0.7). There was no racial disparity in DRFS in the overall cohort or TN subtype, but there was a trend towards worse survival in Black women in the ER+/HER2- subtype (Log-Rank p=0.09). TMEM doorway density was higher in Black women in the entire cohort (p=0.001) and in the ER+/HER2- subtype (p=0.01), while there was no difference in the TN subtype. TMEM doorway density was prognostic for DRFS in all patients (Log-Rank p=0.009) and the ER+/HER2- subtype (Log-Rank p=0.001), but not in the TN subtype. There was a trend towards higher BCSC density in the entire cohort (p=0.06) and the ER+/HER2- subtype (p=0.09), but there was no difference in the TN subtype. There was no racial disparity in motile cell density in the entire cohort as well as in the two subtypes. BCSC density and motile cell density were not prognostic for DRFS. Conclusion: Worse DRFS in Black, compared to White, women with ER-positive residual breast cancer post-NAC may be due to differences in TMEM doorway and BCSC density. Further studies are necessary to determine if the outcome of patients with residual disease and high TMEM doorway density post-NAC could be improved by blocking TMEM doorway function with TIE2 inhibitors. Citation Format: Gina Kim, Saeed Asiry, Isabelle Oktay, Yu Lin, Xianjun Ye, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D'Alfonso, David Entenberg, Maja Oktay. Racial disparity in post-neoadjuvant chemotherapy residual breast tumor microenvironment [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 P1-08-36.

Even detected in its earliest stages, lung cancer is a deadly disease, with fewer than 50% of cases surviving 5 years post-diagnosis. As surgical resection of the tumor is the extent of treatment for most early stage disease, ways to identify individuals who would benefit from additional therapy is critical to reduce the mortality in this population. Epigenetic changes are an important mechanism in carcinogenic progression, but their potential role in lung cancer progression is not yet understood. We identified formalin fixed, paraffin embedded (FFPE) tumors with high cellularity from 20 white women with early stage adenocarcinoma of the lung and two matched blood DNA samples from a previous population-based case-control study. These samples were profiled on the Infinium HumanMethylation450 BeadChip after processing with the Infinium HD FFPE Restore Kit in the University of Michigan Sequencing Core. The HumanMethylation450 BeadChip measures the methylation status at over 480,000 cytosines distributed over the genome. Excluding sites with missing values and high detection p-values, 466,807 sites were available for analysis. The Bioconductor package IMA was used for data processing and the R package limma was used to identify methylation differences based on survival (≥ 36 months, n = 14 vs < 36 months, n=6) and somatic mutation status (2 samples each were either EGFR or KRAS positive) after adjustment for pack years of smoking and age at diagnosis. Comparing those who did and did not survive 36 months post-diagnosis, 2178 probes were differentially methylated after correction for multiple testing (adjusted q<0.20). Of these, 270 (12.4%) were in CpG islands and 1040 (47.8%) were not located in a CpG island, shore or shelf. Comparing those with and without KRAS mutations, 1017 genes were differentially methylated. Of these, 596 (58.6%) were in CpG islands and 157 (15.4%) were not located in a CpG island, shore or shelf. There were fewer differences based on EGFR status, with only six methylation differences significant at q<0.20. Ongoing validation analyses are underway to understand the extent and significance of differentially methylated sites. In this pilot study using a newly-designed, high density microarray for methylation analysis on FFPE samples, we identified a distinct epigenetic prognostic profile in lung cancer. We also note a potential epigenetic profile associated with KRAS mutations in lung adenocarcinomas, as has been noted in other tumor sites. Our initial results indicate specific epigenetic differences may correlate with survival in early stage adenocarcinoma of the lung and could provide insight into functional changes that affect disease progression, and ultimately survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-383. doi:1538-7445.AM2012-LB-383

Background: Biomarker testing in lung cancer is often limited by a lack of sufficient formalin fixed, paraffin embedded (FFPE) tissue for comprehensive genomic profiling. To promote personalized therapy for lung cancer, a multiplex FISH assay was developed to simultaneously assess aberrations in ROS1, RET, and MET on a single FFPE specimen slide. Methods: Specimens included primary tumor (N = 39) as well as biopsies from a variety of metastatic sites (N = 16). These included 12 samples with ROS1 rearrangements, 3 samples with RET rearrangements, and 11 samples with MET amplification reported by a previously validated laboratory test method. A probe mix contained 6 differentially labeled fluorescent probes: 3' ROS1, 5' ROS1, 3' RET, 5' RET, MET and CEP7. The probes were formulated in Vysis IntelliFISH Hybridization Buffer to allow for a 2 h hybridization time. BioView imaging platform and Duet software algorithm were used to perform automated slide scanning and digital analysis. Specimens were considered positive for ROS1 or RET rearrangement if >15% evaluated cells contained a break apart (rearranged) signal. Specimens were considered positive for MET amplification if >15% of cells had MET/CEP7 ratio >2 and positive for polysomy if >15% of cells had 5 or more MET signals copies. Results: The 6 color FISH assay was 97% concordant for ROS1 rearrangement and 100% concordant for RET rearrangement. The average background percentage of positive tumor cells in cases without known gene rearrangements was approximately 5%, yielding a negative cutoff threshold of approximately 15%, in accordance with cutoff thresholds reported in literature. The 6 color FISH assay was 91% concordant for MET amplification or polysomy. Results were interpretable for 98% of targets analyzed by the 6 color FISH method. Four samples failed on analysis for one of the targets due to lack of sufficient cells or lack of adequate hybridization signal. Conclusion: A newly developed 6-color FISH assay allows simultaneous detection of three genomic abnormalities using only 1 specimen slide. This feature combined with rapid hybridization in IntelliFISH buffer and automated BioView slide imaging and analysis can significantly increase the yield of molecular testing on limited lung cancer tissue samples. Careful pathologic correlation for tumor cell identification and careful assessment of hybridization quality are necessary to optimize the accuracy of this test method. Citation Format: Irina A. Sokolova, Patrick Bedroske, Tatyana A. Grushko, Amber R. Schneider, Kristine Jacobson, Jesse S. Voss, Yishay Tauber, Yossi Avisror, Vitaliy Shkolnik, Katerina Pestova, Katherine B. Geiersbach. Multiplex fast FISH assay for detecting ROS1, RET and MET aberrations in FFPE specimens using BioView image analysis [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 4256.

Background: Renal cancers account for more than 3% of adult malignancies and result in more than 13,000 deaths per year in the US alone. The four most common types of kidney tumors include the malignant renal cell carcinomas: clear cell, papillary and chromophobe, as well as the benign oncocytoma. These histological subtypes vary in their clinical course and their prognosis, and different clinical strategies have been developed for their management. The differential diagnosis between the subtypes of kidney tumors based on morphology alone can be challenging, and is subjected to inter- and intra-observer variability. Even when utilizing immunohistochemistry (IHC) markers, the ability to differentiate between sub-types can be difficult, especially in the setting of uncommon morphology and biopsy sample with small amounts of tumor tissue. We present the development and validation of a microRNA-based test for classifying primary kidney tumors. Methods: 181 Formalin Fixed Paraffin Embedded (FFPE) samples from primary kidney tumors were collected and reviewed by pathologists from different institutes according to morphology and available IHC labeling data. High-quality total RNA, including the well-preserved microRNA fraction, was extracted from the FFPE samples using a proprietary protocol. Expression levels of hundreds of microRNAs were profiled using a custom microarray platform. Technical validation of the array results was performed using qRT-PCR. A diagnostic assay was developed using a K-nearest neighbor algorithm that searches for the 5 samples in the training database (181 samples used for assay development) that are most similar to the tested sample. The result for the tested sample is defined by a majority vote of the pre-determined subtypes of these 5 closest neighbors. A validation set of 201 independent samples was classified using the assay and analyzed blindly. Results: A set of 24 differentially expressed microRNAs were found to separate the four kidney tumor subtypes and were chosen as classifiers in the KNN algorithm. Clinical validation was performed using an independent, blinded sample set. The test was able to produce results for 92% of the validation set of 201 samples with accuracy of 95%. Conclusions: Expression levels of 24 microRNAs measured on a microarray platform were found to accurately differentiate the four main types of primary kidney tumors. These findings were the basis for the development and validation of a standardized diagnostic assay for the classification of renal cell tumors in FFPE samples from resections or biopsies. This assay can serve as a reliable diagnostic tool to aid physicians with the growing unmet need for kidney tumor classification. Citation Format: Robert Wassman, Brianna St. Cyr, Eddie Fridman, Iris Barshack, Yajue Huang, Sofia Zilber, Mats Sanden, Hila Benjamin, Noga Yerushalmi, Yael Spector. Development and validation of a microRNA-based diagnostic assay for the classification of renal cell tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2013-802