Formalin-fixed Paraffin-embedded Cell Lines Research Articles

Establishing clinical performance characteristics for Duoseq, a new assay for DNA and RNA sequencing.

e15059 Background: Despite advances in genomics and next generation sequencing (NGS), clinical implementation of NGS remains challenging. This challenge is critical in hematologic malignancies where NGS has become an essential part of the clinical workup. Many clinical labs resort to send-out testing that can delay appropriate treatment and drive up the cost of care. To address these issues, we have developed Duoseq as a unified approach for DNA and RNA NGS. Duoseq assesses mutations (single nucleotide variants [SNVs], insertions/deletions [indels] and splice site variants [SSVs]), structural variants, and gene expression using a panel of over 400 genes. It is supplied as a kit that enables preparation of DNA and RNA sequencing libraries in a single workflow performed at the point-of-care. It includes complete bioinformatics support that enables a clinical laboratory with an NGS instrument to perform the assay with a turn-around time of 2-3 days. Duoseq is optimized to work with formalin-fixed, paraffin embedded (FFPE) samples that are the standard fixation practice. Methods: We evaluated Duoseq on four dimensions critical to clinical performance: reproducibility, precision, analytical sensitivity (limit of detection, LoD), and analytical specificity. Identical experiments were run at clinical laboratories of two different institutions. Analytical validation samples included 4 FFPE cell lines and 2 synthetic DNA samples designed to harbor variants of interest at known allele frequencies. Results: We determined the LoD to be 5% mutant allele fraction (MAF) for SNVs, 10% MAF for indels/SSVs, and 10% tumor purity for SVs. Duoseq achieved 100% precision and reproducibility for each variant class for events at or above the LoD. Analytical specificity was 100%, indicating no instances of cross-contamination of samples within or across runs. We further evaluated Duoseq performance in 54 clinical cases of myeloid and lymphoid malignancies compared against clinical results provided by FoundationOne Heme NGS panel. Collectively, we assessed 119 SNVs and 49 indels/SSVs. Duoseq performed with 100% specificity for both SNVs and indels/SSVs. Duoseq achieved 98% sensitivity for SNVs and 96% for indels/SSVs. For example, Duoseq detected TP53 variants in 6 cases of myeloid neoplasms, which has relevance for the 2022 WHO and ICC classifications. Duoseq also identified therapeutically relevant variants in IDH1/2, EZH2, FLT3, KRAS and TET2. Conclusions: This study demonstrates performance characteristics that suggest Duoseq would perform well as a clinical-grade assay. Local laboratory factors that include staffing, case volumes, and reimbursement affect adoption and turnaround times in the individual clinical laboratory. However, we anticipate that Duoseq implementation will accelerate the diagnosis of hematologic malignancies and facilitate rapid, more cost-effective care to improve patient outcomes.

Abstract 1613: Enhanced sensitivity HERmark® assay: A sensitive and quantitative immunoassay superior to immunohistochemistry for the measurement of low HER2 protein levels in formalin-fixed, paraffin-embedded samples

Abstract Introduction: Breast cancers that express low levels of HER2 that are not considered HER2-positive disease by current HER2 classification standards are the targeted treatment populations for many new and promising HER2-targeted therapies, e.g. HER2 antibody-drug conjugates, tyrosine kinase inhibitors and vaccines. HER2 status is routinely determined by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These technologies may have limited utility or lack the sensitivity required to adequately stratify low HER2 protein expression. Following upon the successes of the HERmark Breast Cancer Assay, we have developed the Enhanced Sensitivity HERmark Assay (ESHA) to quantify low HER2 protein expression in formalin-fixed, paraffin-embedded (FFPE) samples. The ESHA format relies on dual-antibody binding for the proximity-dependent release of a fluorescent reporter, which is measured with high sensitivity and reproducibility via capillary electrophoresis to accurately quantify HER2 protein expression. Methods: The HERmark and HERmark ESHA assays utilize HER2 mAb8 conjugated to a light-releasable VeraTag® reporter and HER2 mAb15 conjugated to a photosensitizer. Low HER2 expressing cancer cell lines were used to optimize assay conditions that improved signal strength and extended the lower end of the dynamic range of the HERmark assay. FFPE cancer cell lines and breast tumors were utilized to evaluate assay background, reproducibility, and to compare HER2 assessments based on HERmark, HERmark ESHA and HER2 IHC. Results: The ESHA format expanded the HERmark assay lower limit of quantification by more than 10-fold, and increased the relative fluorescent signal by an average of 16-fold, which also resulted in a decrease in assay variation. ESHA measurements of HER2 protein in a panel of FFPE cell lines correlated with HER2 gene expression levels as reported in the cancer cell line encyclopedia (https://portals.broadinstitute.org/ccle, R2 = 0.857). ESHA measurements of HER2 protein correlated well with conventional IHC scores, along with the added ability to quantitatively distinguish HER2 protein expression levels. Conclusions: We have developed an Enhanced Sensitivity HERmark Assay for the quantification of low HER2 protein expression in formalin-fixed, paraffin-embedded tissue samples. This assay can be a particularly useful tool for the development of novel HER2-targeting therapies in patient populations that are not considered HER2 positive by conventional HER2 classification standards. Improving the stratification of HER2 expression may better define, and perhaps expand, the number of patients that are more likely to benefit from HER2-targeted therapy. Citation Format: Gerald Wallweber, Roy Ravanera, Paul Theobald, Weidong Huang, Christos Petropoulos. Enhanced sensitivity HERmark® assay: A sensitive and quantitative immunoassay superior to immunohistochemistry for the measurement of low HER2 protein levels in formalin-fixed, paraffin-embedded 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 1613.

Abstract 1383: NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues

Abstract Background: HTG Molecular (HTG) developed a targeted Next Generation Sequencing (NGS)-based gene expression assay that measures mRNA levels of 2,560 genes (2,532 oncology-related biomarker genes). The HTG EdgeSeq Oncology Biomarker Panel Assay (OBP) is based on a novel derivative of our quantitative nuclease protection chemistry (qNPA) that enables extraction-free quantitation (detection) of mRNA from variety of sample types including formalin-fixed, paraffin embedded (FFPE) tissue. Purpose: To determine (1) linearity across a range of sample inputs, (2) recommended sample input amounts for FFPE, cells, and extracted RNA, and (3) reproducibility of the HTG EdgeSeq Oncology Biomarker Panel Assay in measuring the mRNA expression of 2,560 genes. Methods: Lysates of 5 micron sections of FFPE tissues (lung, breast, prostate, and colon carcinoma; melanoma; 25 mm2 to 0.78 mm2 per reaction), THP-1 and HCC78 cell lines (7500 cells to 234 cells per reaction), and Universal RNA (URNA; 25 ng to 1.56 ng per reaction) were used for the linearity and sample input studies. URNA (25 ng per reaction) was used to demonstrate reproducibility of the assay across multiple days and processors (one processor across three days and three processors on one day). Sequencing libraries were generated from the qNPA reactions and run on an Illumina MiSeq sequencing platform. The HTG EdgeSeq Parser was used for post-sequencing data processing. Linear regression (R2) and Pearson correlation coefficients (r) were used to assess linearity and reproducibility of the assay. Results: The R2 for linearity across four concentration points for lung FFPE tissue (6.25-0.78 mm2), cell lines (1875-234 cells), and URNA (12.5-1.56 ng) were >0.97, 0.99, and 0.99, respectively. The (r) between low (1.56 mm2) and high (12.5 mm2) sample inputs for each FFPE tissue type was > 0.98. The (r) for intra-run, inter-day, and inter-run reproducibility were > 0.95, > 0.98, and > 0.98 respectively. In addition, differential expression of tissue-specific genes was identified in the respective FFPE tissues, including NKX2 and MUC1 in lung, ERBB2 in breast, NKX3, KLK2, and KLK3 in prostate, and SPP1 and PRAME in melanoma. Conclusions: The HTG EdgeSeq Oncology Biomarker Panel Assay for a 2,560-gene panel of oncology-related biomarkers is linear over a wide range of sample inputs, can comprehensively analyze very small, clinically relevant tissues, and is highly reproducible. The demonstrated performance of the assay in breast, lung, colon, and prostate cancer and melanoma FFPE samples enables multiplex oncology biomarker profiling of these and other malignant neoplasms. Citation Format: Monica M. Reinholz, Debrah M. Thompson, Ihab Botros, Matt Rounseville, Patrick C. Roche. NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1383.

Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR

ObjectiveTo validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of...

Abstract LB-B18: Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy

Abstract Epithelial-mesenchymal transition (EMT) is a dynamic process whereby epithelial cells acquire mesenchymal properties. Despite the clinical significance of the acquired mesenchymal properties for metastasis and drug resistance, histopathological evidence of transitional cells in patient samples is lacking and EMT remains an unproven clinical hypothesis. We previously developed and validated a multiplex immunofluorescence assay (IFA) that quantifies the levels of EMT biomarkers (E-Cadherin, Vimentin, β-catenin) in snap-frozen, formalin-fixed, paraffin-embedded (FFPE) tumor tissue (Navas et al, AACR 2013). Building upon that method, we now report a precise, quantitative and unbiased IFA method of tissue analysis (EMT-IFA) using Definiens® software to quantify co-expression of the epithelial marker E-cadherin (E) and mesenchymal marker Vimentin (V) at the cellular level in FFPE clinical biopsies. FFPE human tumor xenografts and cell lines serve as calibrators and reference materials for establishing initial image acquisition parameters for segmented tumor regions of interest. Flanking H&E sections of the clinical biopsies are initially annotated by an anatomic pathologist to identify regions of tumor tissue, non-tumor related areas (including normal or stromal tissues), inflammatory and/or necrotic regions, and freeze artifacts. Adjacent sections are used for the EMT-IFA, and further tumor segmentation from stroma is applied to extracted regions of interests (fields) using the β-catenin layer. Within segmented tumor regions, E-cadherin (E) and Vimentin (V) sub-cellular pixel areas are measured using predetermined thresholds for each biomarker and reported as Log10 (V:E). High resolution images confirmed the co-localization of plasma membrane E-cadherin and cytoplasmic Vimentin to individual transitional tumor cells, and the co-localization of E and V within individual tumor cells of the segmented regions of interest was key for distinguishing EMT from tumor heterogeneity in which adjacent tumor cells can be exclusively E+ or V+. Quantitative analysis of a clinical biopsy series of various histologies revealed all possible phenotypes: epithelial (E+V- colorectal carcinoma), mesenchymal (E-V+ sarcomas), heterogeneous mixtures of E+V- and E-V+ subpopulations, and EMT. Importantly, using the EMT-IFA, we discovered that pharmacological targeting of VEGFR/FGFR/PDGFR signaling with the multi-kinase inhibitor pazopanib stimulated EMT in a preclinical xenograft model of gastric carcinoma (MKN45), as revealed by the significant increase in the V:E ratio (P = 0.0023) coupled with an increase in the EMT phenotype (co-localized V+E+ at the individual cell level) (P = 0.0070) in biopsy specimens. Thus, the EMT-IFA has potential value for investigating EMT in the clinic and its ramifications for drug response and other clinical endpoints. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Robert J. Kinders, Scott M. Lawrence, Katherine Ferry-Galow, Thomas D. Pfister, Apurva K. Srivastava, Sergio Y. Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna O. Butcher, Hala Makhlouf, Rodrigo Chuaqui, Donald P. Bottaro, Shivaani Kummar, Alice Chen, James H. Doroshow, Ralph E. Parchment. Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B18.

Abstract A1-43: Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations

Abstract Background: Although precision medicine approaches have revolutionized oncology, widespread adoption requires robust, inexpensive approaches enabling the targeted assessment of all relevant alteration classes from routine tissue samples. Methods: Here we interrogated >7,000 cancer exomes and transcriptomes, along with >30,000 array based cancer genomes to identify recurrent somatic alterations (mutations, copy number alterations [CNAs] and gene fusions) across solid tumors. From this analysis, we developed and validated an integrated multiplexed PCR based Ion Torrent next generation sequencing panel (Oncomine Cancer Research Panel [OCP]) targeting the actionable somatic cancer genome optimized for 20ng of formalin-fixed, paraffin-embedded (FFPE) tissue isolated DNA/RNA. Results: We validated the OCP using FFPE cell line mixtures, as well as a prospective cohort of 104 FFPE tumor specimens sent for concurrent clinical molecular testing, with >97% sensitivity and specificity for the presence/absence of KRAS, EGFR, BRAF and ALK point mutations, indels or gene fusions in this molecular testing cohort. We also applied the OCP to 100 lung cancers, identifying known and novel alterations, including ALK and ROS1 gene fusions. Lastly, applying the OCP to 116 prostate cancers, including 50 previously treated samples, we recapitulated known molecular subtypes, observed distinct profiles according to previous treatment and obtained 100% concordance for isoform specific TMPRSS2:ERG gene fusion detection compared to qPCR. Additionally, OCP profiling supports a novel molecular subtype of prostate cancer defined by IDH1 R132 hotspot mutations and informed on resistance mechanisms in a pre- and post-treatment sample pair. Importantly, 44%, 35% and 9% of patients in the molecular testing, lung and prostate cancer cohorts, respectively, harbored additional alterations (beyond routine molecular testing) associated with FDA approved or NCCN guideline referenced therapies. Conclusions: Through analysis of both DNA and RNA to assess the actionable somatic cancer genome, the validated OCP panel may have utility in both clinical and research settings. Citation Format: Daniel H. Hovelson, Andrew S. McDaniel, Bryan Johnson, Andi K. Cani, Kate Rhodes, Paul D. Williams, Chia-Jen Liu, Santhoshi Bandla, Catherine S. Grasso, Michael J. Quist, Seth Sadis, Daniel R. Rhodes, Scott A. Tomlins. Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-43.

Abstract 1892: Development of a clinical targeted next generation sequencing test for challenging formalin-fixed paraffin-embedded (FFPE) cancer samples

Abstract Introduction: There is an urgent need for deep targeted sequencing for detection of varieties of somatic aberrations, including single nucleotide variants (SNV), insertions/deletions (indels), loss of heterozygosity (LOH), and copy number variants (CNV) in heterogeneous FFPE cancer patient specimens. We have developed a Next Generation Sequencing (NGS)-based assay for deep sequencing of a number of selected oncogenes and tumor suppressor genes for testing of clinical breast cancer samples. The panel includes those genes, which are commonly mutated in cancers (e.g. breast and ovarian cancer), based on The Cancer Genome Atlas studies and are potentially indicative of responses to experimental compounds undergoing clinical trials. Importantly, the assay can be used in FFPE samples which pose challenges because of high variability in quality and often suboptimal quantities. Methods: To minimize FFPE sample input requirement, we optimized multiple assay steps. DNA was extracted from macrodissected FFPE tissue, and randomly fragmented. The DNA library was constructed and subject to target capture and enrichment before being sequenced on the Illumina MiSeq® systems. To analytically qualify the assays, we tested well-characterized cell lines and reference HapMap cell lines. Furthermore, to confirm the performance of the NGS results in clinical FFPE samples, we compared the results generated using orthogonal methodologies, e.g. SNVs using a custom Ion AmpliSeq™ panel (Life Technologies) on Ion Personal Genome Machine® (PGM), or CNVs using real-time PCR assays or custom Nanostring nCounter® CNV assays (NanoString Technologies). To establish the lower limit of detection, we used mixed DNA samples from FFPE cell lines and from HapMap DNA and further tested Horizon FFPE reference DNA containing mutations at defined allele frequencies. Results: For FFPE DNA extraction, we selected Cobas® FFPE kit (Roche), and Maxwell® CSC DNA FFPE kit (Promega) for higher yield and better purity. For library construction, we chose a KAPA streamlined library preparation kit. For target capture, we used custom xGEN® DNA probes (Integrated DNA Technologies) for improved flexibility in assay optimization, reduced reagent cost, and minimizing batch-to-batch variability. We were able to detect SNVs for >99% of exons selected down to a 5% sensitivity level using about 100 ng FFPE DNA input. Indels, CNV and LOH could also be detected reliably. Conclusions: We developed a targeted NGS assay that could be used in clinical trials and meets the dual challenge of limited DNA amounts and/or variable quality often associated with FFPE clinical specimens and the detection of low level mutations. Considering the recent recognition of Illumina NGS platform by FDA, this approach can represent a path forward to assist in cancer patient assessment, clinical trial recruitment and care. Citation Format: Wenge Shi, Christine Chin, Tingdong Tang, Loretta Hipolito, Preethi Srinivasan, Derek Chiang, David Peng, Emmanuelle Di Tomaso, Shabnam Tangri, Jelveh Lameh, Reinhold Pollner. Development of a clinical targeted next generation sequencing test for challenging formalin-fixed paraffin-embedded (FFPE) cancer samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2014-1892

PD-L1 expression in non-clear cell renal cell carcinoma.

424 Background: Programmed death-1 (PD-1) receptor negatively regulates T cell-mediated responses.PD-1 ligand (PD-L1) is aberrantly expressed in clear cell renal cell carcinoma (ccRCC) and is associated with worse prognosis. Levels of PD-L1 expressions in non-ccRCC and its association with clinicopathological features and survival are unknown. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 97 patients with chromophobe (CHR), papillary (PAP), translocation Xp11.2 (TrL) RCC and oncoytoma (ONC) and were included in the analysis. PD-L1 expression was evaluated by immunohistochemistry using a mouse monoclonal anti-PD-L1 antibody (405.9A11). The assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry. PD-L1 tumor positivity (PD-L1+) was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in immune cells, a combined score based on the intensity of infiltrate and percentage of positive cells was used. Baseline characteristics including stage/grade, and survival data were collected. Comparisons between PD-L1 expression and clinicopathological features were evaluated using chisq or wilcoxon rank sum tests. Cox model tests for association of PD-L1 expression with OS in univariate and multivariable analysis. Results: Among 97 patients, 12 (12.4%) were considered PD-L1+ in tumor cells: 2/36 (5%) of CHR RCC, 5/50 (10%) of PAP RCC, 3/7 (43%) of TrL RCC, and 2/4 (50%) of ONC. PD-L1 positivity in tumor cells was significantly associated with higher stage (p=0.026) and grade (p=0.046), as well as lower OS on univariate (p=0.02) but not multivariate analysis (p=0.29). On the other hand, PD-L1 positivity by immune cells was observed in 50 (51.5%) patients: 13/36 (36%) of CHR RCC, 30/50 (60%) of PAP RCC, 6/7 (86%) of TrL RCC, and 1/4 (25%) of ONC. PD-L1 positivity in immune cells did not correlate with stage (p=0.7), grade (p=0.1) or OS (p=0.8). Conclusions: PD-L1 expression is variable in non-ccRCC and depends on histology and tumor vs. immune cells scoring. Only PD-L1 positivity in tumors cells was associated with aggressive features. Patients with non-ccRCC should not be automatically excluded from trials of agents that target the PD-1/PD-L1 pathway.

PD-L1 expression in primary clear cell renal cell carcinomas (ccRCCs) and their metastases.

467 Background: Clinical trials evaluating anti-PD-1 and anti-PD-L1 antibodies (Abs) in ccRCC have shown promising efficacy in a subset of patients. Preliminary studies have demonstrated that tumor PD-L1 expression increases the likelihood of benefit with anti-PD-1 Ab, but fails to identify all responders. One potential explanation for these results is that predictive biomarkers are usually evaluated in the primary tumors, which are more readily available; however, biomarker expression in nephrectomy samples may not accurately reflect expression in the metastases that are being targeted by therapy. In this study, we compared PD-L1 expression in a series of ccRCCs and their metastases. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue blocks from 34 primary ccRCCs and corresponding metastases were retrieved. Multiple areas of the primary tumors, including areas of predominant and highest Fuhrman nuclear grade (FNG), were selected for analysis. Slides were immunostained with a mouse monoclonal anti-PD-L1 antibody (405.9A11). The assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry. The presence of tumor cells with membranous staining was assessed. A case was considered positive when any tumor cell positivity was detected. Results: Positive membranous PD-L1 expression in tumor cells was observed in 10/34 (29%) primary ccRCCs. In 3 of these 10 cases (30%), the metastases were negative. In 2 cases the primary tumor was negative but the metastases were positive. In twenty-two cases, both the primary tumor and the corresponding metastasis were negative. The pattern of PD-L1 staining was highly heterogeneous in the primary tumors and was restricted to areas of highest FNG. The staining was more homogeneous in the metastases. PD-L1 expression by the tumor infiltrating immune cells is currently being evaluated. Conclusions: Discordant expression of PD-L1 between the primary tumor and the corresponding metastases was detected in 5/34 (15%) cases, suggesting that accurate assessment of predictive biomarkers for PD-1 blockade in ccRCC might require analysis of metastatic lesions. Analysis of a larger patient cohort is ongoing to confirm these findings.

Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Assessment of tissue distribution and subcellular localization of miR-302 and miR-21 by means of in situ hybridization technique

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular processes, and their disregulations have been linked to several pathologic conditions, mainly cancers. Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs (miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks, proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection in FFPE samples and NT2 cell line.

Abstract P3-06-25: PTEN mRNA positivity using in situ measurements is associated with better outcome in Tamoxifen treated breast cancer patients.

Abstract Background: Aberrant phosphatidylinositol 3-kinase (PI3K) pathway signaling occurs in a high proportion of human breast cancers and is involved in carcinogenesis, tumor progression and resistance to targeted therapies. Reduced levels of the tumor suppressor PTEN has been associated with PI3K pathway activation and is observed in ∼40% of breast carcinomas. PTEN levels have traditionally been determined in breast tumors by qualitative immunohistochemistry (IHC) and its prognostic and/or predictive value has not been clearly established. We used a novel method of quantitative in situ measurements of PTEN mRNA and protein to determine their possible prognostic and predictive value in breast cancer. Materials and methods: PTEN mRNA and protein were measured on formalin fixed paraffin embedded (FFPE) cell lines and tumor samples on tissue microarrays (TMAs) representing a retrospective cohort of 404 breast cancer cases collected from the Yale Pathology archives between 1975 and 2005. The RNAscope paired-primer method was used for PTEN mRNA in situ hybridization (ISH), with Ubiquitin C and DapB ISH probes used as positive and negative controls for each run. PTEN protein measurements were performed with monoclonal antibody 138G6 (Cell Signaling Technology). Both mRNA and protein were measured using the AQUA method of quantitative immunofluorescence. Results: Both PTEN mRNA ISH signal and protein were highly reproducible in FFPE tissues and cell lines. PTEN mRNA levels were lower in cell lines with known PTEN downregulation. In breast carcinoma samples, PTEN mRNA scores showed a positive non-linear association with protein levels. Neither PTEN mRNA nor protein showed prognostic value. However, high PTEN mRNA expression using a visually defined cutoff was associated with better outcome in a subset of Tamoxifen treated breast cancer patients. Conclusion: Initial data suggests that PTEN mRNA expression may be predictive of favorable response to endocrine therapy in breast carcinomas. However, validation in larger independent cohorts is required. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-25.

Abstract 3071: Accurate and sensitive detection of KRAS mutations in heterogeneous cancer specimens

Abstract The discovery of pivotal genetic alterations and the understanding of their role in cancer is leading to remarkable successes in therapeutics and patient care. Molecular diagnosis methods such as DNA sequencing and conventional genotyping of tumor biopsies have advanced research in this field, but are limited in sensitivity due to stromal contamination and by genetic heterogeneity in cancer. We have recently developed competitive allele specific TaqMan® PCR (castPCR) assays assays for detecting cancer-associated sequence variations. CastPCR not only maintains the wide dynamic range, high sensitivity and reproducibility of TaqMan® assays but also greatly improves the specificity. The technology enables detection, of as little as 1 mutant allele molecule in 10,000,000 wild type molecules. We report here sensitive and accurate detection of cancer-associated KRAS mutations within formalin-fixed paraffin-embedded (FFPE) heterogeneous cancer specimens. Eight FFPE model cell lines were initially used to validate the assays (NCI-H2009:p.G12A; SW1463:p.G12C; PANC-1:p.G12D; PSN-1: p.G12R; A549:p.G12S; SW480:p.G12V; DLD-1:p.G13D; Jurkat:Wild Type). Mutant FFPE cell line DNAs were titrated in the FFPE wild type cell line DNAs from 100% to 0.1%. Mutations were easily identified at the level of 0.1% with high reproducibility. 24 anonymous tumor tissues and 12 non-tumor tissues from FFPE specimens were also examined. No positive samples were found in non-tumor tissues. The results obtained by castPCR for the 24 tumor tissues were concordant to those previously reported by three different methods (Taqman® PCR, Taqman® PCR + PNA and Sequencing). Our results demonstrate that castPCR, as a new rare mutation detection technology, has greater sensitivity, specificity and can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2011-3071

Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels ofc-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens.

Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens

BackgroundColorectal cancer patients harbouring KRAS mutations in codon 12 or 13 do not benefit from current anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. Efficient and robust methods are therefore...

Abstract B177: Detection and quantification of ligand-receptor interactions in formalin-fixed paraffin embedded samples by a novel proximity-based assay

Abstract Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) sections, a format used to preserve human tissue samples. This is the only format for readily available tissues from clinical trials and patient disease management. However, phosphorylation is generally less stable and consequently, less reproducible which can, in a clinical setting lead to ambiguous data. Here, we describe an alternative, novel approach to detect and quantify activation of receptors in FFPE samples. We used the ligand/receptor, HGF/c-MET as our model system. HGF is the only known ligand of c-MET and ligand binding causes activation of the c-MET receptor. Using, the VeraTag technology, a proximity-based assay, validated antibodies were used to detect and quantify total c-MET and total HGF in FFPE samples as well as to detect and quantify ligand stimulated and endogenous HGF/c-MET interactions in FFPE cell line pellets. The detection was validated in human non-small cell lung and stomach carcinoma FFPE samples. Our data demonstrate a novel approach to detect and quantify ligand-receptor interactions by VeraTag proximity-based assays that can be widely applied to measure activation of receptors in archived FFPE human tissue samples. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B177.

MET, HGF, EGFR, and PXN Gene Copy Number in Lung Cancer Using DNA Extracts from FFPE Archival Samples and Prognostic Significance

Gene copy number analysis for some of the important molecules in lung tumorogenesis, such as MET, hepatocyte growth factor [(HGF), ligand for MET), epidermal growth factor receptor (EGFR), and paxillin (PXN), is likely to determine both the type of treatment and prognosis. Formalin-fixed paraffin-embedded (FFPE) archival tumor tissue samples are an excellent source for determining key molecular changes in the OncoGenome; however, existing extraction procedures yield relatively poor quality genomic DNA fragments. Although FISH is the method of choice for determining amplification of a gene, a more rapid quantitative poly-merase chain reaction (qPCR) technique to determine gene copy number can be used when reasonably good quality genomic DNA is available. We report here a relatively rapid method based on microwave/chelex-100 treatment that gives rise to genomic DNA fragments ranging from 1 to 12 Kb and beyond, thereby attesting to its superior quality. Genomic PCR for beta-globin gene gave reliable and reproducible results. The number of steps for extracting the DNA was kept to a minimum, and instead of precipitating the DNA, we preserved the genomic DNA extracts so as to prevent a loss in DNA yield. We found the extracts to be stable and amenable to qPCR and mutational analysis. Using lung adenocarcinoma FFPE samples and cell lines derived from lung adenocarcinomas, we demonstrated that the gene copy number for MET in lung adenocarcinoma tissue samples was preferentially increased over EGFR, HGF, and PXN and that it positively correlated with a better prognosis. In contrast, the genomic DNA extracted from 25 NSCLC cell lines gave a relatively higher gene copy number for all four genes evaluated. Our results indicate that the microwave/chelex-100-based method yields good-quality genomic DNA extracts that can be used for complex DNA analysis, such as determination of gene copy number. In addition, our data demonstrated that the adenocarcinoma cell lines potentially evolved under ex vivo conditions, and therefore, in genetic studies it is imperative to use primary tumors for generalized conclusions about lung tumors.