Cancer Tissue Sections Research Articles

Immunohistochemistry Microarrays.

Immunohistochemistry (IHC) on tissue sections is widely used for quantifying the expression patterns of proteins and is part of the standard of care for cancer diagnosis and prognosis, but is limited to staining a single protein per tissue. Tissue microarray and microfluidics staining methods have emerged as powerful high throughput techniques, but they either only permit the analysis of a single protein per slide or require complex instrumentation and expertise while only staining isolated areas. Here, we introduce IHC microarrays (IHCμA) for multiplexed staining of intact tissues with preserved histological and spatial information. Droplets of a dextran solution containing antibodies were prespotted on a slide and snapped onto a preprocessed formalin-fixed, paraffin-embedded (FFPE) tissue section soaked in a polyethylene glycol solution. The antibodies are confined within the dextran droplets and locally stain the tissue below with a contrast similar to the one obtained by conventional IHC. The microarray of antibody droplets can be prespotted on a slide and stored, thus neither the preparation of the antibody solutions nor a sophisticated microarray spotter is needed. Sampling considerations with IHCμA were evaluated by taking three tissues with varying levels of cancer cells. A multiplex IHCμA with 180 spots targeting 8 cancer proteins was performed on a breast cancer tissue section to illustrate the potential of this method. This work opens the avenue of applying microarray technologies for conducting IHC on intact tissue slices and has great potential to be used in the discovery and validation of tissue biomarkers in human tumors.

Elevated Hu-Antigen Receptor (HuR) Expression is Associated with Tumor Aggressiveness and Poor Prognosis but not with COX-2 Expression in Invasive Breast Carcinoma Patients.

Hu-antigen R (HuR), a RNA-binding protein, is considered to play a crucial role in tumor development and progression by stabilizing or regulating a group of cellular mRNAs of cancer-related genes, such as cyclooxygenase-2 (COX-2). The present study aimed to evaluate the clinical significance of HuR and COX-2 expression in invasive breast carcinoma. HuR and COX-2 protein expression was assessed immunohistochemically on paraffin-embedded breast cancer tissue sections obtained from 121 patients and was statistically analyzed with clinicopathological parameters, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), as well as with tumor cells' proliferative capacity and overall and disease-free patients' survival. High HuR expression was positively associated with larger tumor size and advanced disease stage (p=0.0234 and p=0.0361, respectively), being more frequently observed in ER negative cases (p=0.0208). High COX-2 expression was negatively associated with histological (p<0.0001) and nuclear (p=0.0033) grade and tumor cells' proliferative rate (p=0.0015), being more frequently observed in luminal-A compared to other molecular subtypes (p=0.0221). High HuR expression was associated with poor overall and disease-free patients' survival at both univariate (log-rank test, p=0.0092 and p=0.0004, respectively) and multivariate (Cox-regression analysis, p=0.0223 and p=0.0004, respectively) level. On the other hand, high COX-2 expression was associated with favorable overall and disease-free patients' survival merely at univariate level (log-rank test, p=0.0389 and p=0.0154, respectively). HuR expression was not associated with COX-2 expression (Spearman R=0.1489, p=0.1032). The present data support evidence that HuR is associated with tumor aggressiveness and poor prognosis in breast carcinoma, reinforcing its potential as promising therapeutic target in this type of neoplasia.

Multiplexed ion beam imaging analysis for quantitation of protein expresssion in cancer tissue sections

Part of developing therapeutics is the need to identify patients who will respond to treatment. For HER2-targeted therapies, such as trastuzumab, the expression level of HER2 is used to identify patients likely to receive benefit from therapy. Currently, chromogenic immunohistochemistry on patient tumor tissue is one of the methodologies used to assess the expression level of HER2 to determine eligibility for trastuzumab. However, chromogenic staining is fraught with serious drawbacks that influence scoring, which is additionally flawed due to the subjective nature of human/pathologist bias. Thus, to advance drug development and precision medicine, there is a need to develop technologies that are more objective and quantitative through the collection and integration of larger data sets. In proof of concept experiments, we show multiplexed ion beam imaging (MIBI), a novel imaging technology, can quantitate HER2 expression on breast carcinoma tissue with known HER2 status and those values correlate with pathologist-determined IHC scores. The same type of quantitative analysis using the mean pixel value of five individual cells and total pixel count of the entire image was extended to a blinded study of breast carcinoma samples of unknown HER2 scores. Here, a strong correlation between quantitation of HER2 by MIBI analysis and pathologist-derived HER2 IHC score was identified. In addition, a comparison between MIBI analysis and immunofluorescence-based automated quantitative analysis (AQUA) technology, an industry-accepted quantitation system, showed strong correlation in the same blind study. Further comparison of the two systems determined MIBI was comparable to AQUA analysis when evaluated against pathologist-determined scores. Using HER2 as a model, these data show MIBI analysis can quantitate protein expression with greater sensitivity and objectivity compared to standard pathologist interpretation, demonstrating its potential as a technology capable of advancing cancer and patient diagnostics.

Abstract 4642: Ion mobility separation of N-Glycans directly from ffpe colon cancer tissue section in a MALDI imaging experiment

Abstract Colon cancer is currently the third most common cancer in both men and women. Research studies have reported extensive alterations in protein glycosylation patterns in cancer tissues. However during these studies, the tissues are homogenized and the spatial information showing the localization of the glycans is lost. Mass spectrometry imaging (MSI) is an established analytical tool for biomolecular research which can accurately determine the spatial location of molecules in a tissue section. Recently, methods have been developed to determine released N-Glycans directly from tissues. A major challenge in the analysis of N-glycans is the large number of isobaric glycans resulting from their complex structures with branched chains and multiple additions residues. Here we report the advantage of ion mobility separation to differentiate these glycans in a MALDI MSI workflow used in the analysis of human FFPE colon cancer tissue. 5µm FFPE tissue were were deparaffinized in xylene, rehydrated in different composition of ethanol/water solutions, transferred in citraconic anhydride buffer for antigen retrieval and water washed. To release the glycans from their proteins, a PNGaseF solution was sprayed on the tissue. A solution of MALDI was sprayed onto the tissue. Experiments were carried out on a SYNAPT G2-Si HDMS system(Waters) where the tri-wave separated ions according to their ion mobility in the gas phase. The overall MALDI spectrum shows strong signal for N-glycan molecules, demonstrating the efficacy of the digestion step of the methodology. Using prior knowledge of the type of glycans, 76 glycans were identified and mapped directly from the FFPE tissue section, from a mass to charge ratio (m/z) of 771.5 up to 2905.03 using accurate mass information. MALDI MSI was able to distinguish the tissue morphology and determine the tumor region based on specific ions, especially sodiated N-Glycans Hex7HexNAc2 (m/z 1581.5) which was highly abundant in the tumor tissue. When the IMS was explored using the DriftScope software, it could clearly been observed that two nested trendlines in m/z vs. DriftTime existed. The faster trendline corresponding to more compact conformation of the ions in the gas phase was identified to be the N-Glycan class of molecules. Using IMS the MS data showed a clear picture of the N-Glycans compare to the MS data alone. In several cases, the IMS peaks were broader than the expected resolution or there were shoulder peaks, indicating that isobaric species were present. For example, m/z 1444.6 ( Hex4dHex1HexNAc3,Na+) displayed two IMS distinct peaks which while not baseline separated were sufficiently separated to obtain individual ion images which showed significantly different distributions. The isobaric species with the faster drift time was evenly distributed across the tissue type of the section, whereas the isobaric species that had the slower drift time was less abundant in the cancer tissue. Citation Format: Emmanuelle Claude, Peggi M. Angel, Richard R. Drake, Hernando Olivos, James I. Langridge. Ion mobility separation of N-Glycans directly from ffpe colon cancer tissue section in a MALDI imaging experiment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4642. doi:10.1158/1538-7445.AM2017-4642

Prognostic value of NK and T-lymphocytes markers in operable non-small cell lung cancer (NSCLC).

11556 Background: Therapies aimed at activation of T and NK cells are developed to expand NSCLC treatments options. It is conceivable that markers of ‘immune ignorant’, ‘immune excluding’ or ‘inflamed’ tumor phenotypes could be prognostic or predictive of benefit from specific immune-targeting therapies. Aim: To assess the prognostic value of expression of T and NK cells mRNA markers and immune-related genes in early stage NSCLC. Methods: qRT-PCR was used to assess 48 mRNAs levels in frozen cancer tissue sections and matched normal lung parenchyma from 56 surgically treated stage I-IIIA NSCLC patients. The mRNA expression (normalized vs. 4 reference genes) was compared between the groups that did (44%) or did not relapse, as well as clinicopathological features (33% never-smokers, 75% lung adenocarcinoma). Results: Low expression of FAS-L (p.adj. = 0.048) , TIGIT and LAG3 was correlated with shorter distant metastasis free survival (DMFS) (p &lt; 0.04). Expression of PD-1 (p = 0.024) and CTLA4 (p = 0.04) was significantly lower in relapsed vs. non-relapsed NSCLCs, whereas there was no difference for PDL-1 and PDL-2. Expression of NK activation markers: NCR3 and NCR1, but not NCR3-ligand 1 was significantly lower in relapsed vs. not relapsed NSCLCs. Other NK cell markers: CD96 and NKG2D were expressed at lower levels (p = 0.02) in relapsed vs. not relapsed NSCLCs, whereas there was no difference for NKG2C and NKG2A. Expression of CXCR3 was lower in relapsed NSCLCs (p = 0.03), however, the expression of its ligands (chemoattractants for lymphocytes) - CXCL9, CXCL10 or CXCL11 or endothelin receptor type B was not different according to metastatic status. GITR and FOXP3 expression was significantly higher in cancers vs. normal lung parenchyma (p.adj. &lt; 0.003). There were no differences in expression according to gender, smoking or NSCLC histological types. Conclusions: Non-inflamed NSCLC phenotype is associated with higher risk of dissemination after primary resection. Neoplastic tissue is characterized by higher level of immune tolerance in comparison to normal lung tissue.

Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas

Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d=6.33±0.86nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490nm wavelength) in the dynamic range of 100-700nM ECD_Apt1 with a detection limit of 12.5±2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.

Inducing Polyclonal Eag1-Specific Antibodies by Vaccination with a Linear Epitope Immunogen and Its Relation to Breast Tumorigenesis.

Ether à-go-go 1 (KCNH1, Kv10.1) (Eag1) is a voltage-gated potassium channel, which is commonly overexpressed in tested breast cancer patients. This occurrence makes it a potential molecular marker and a promising tool for breast cancer diagnosis and therapy. In order to explore protective or specific polyclonal antibodies for further research, potential linear epitopes from Eag1 were collected by sequence alignment. The sequence was synthesized and then coupled to the carrier protein keyhole limpet hemocyanin (KLH) for animal immunization. Polyclonal antibodies against Eag1 were produced and purified from the rabbit antisera. Enzyme linked immunosorbent assay (ELISA) and western blot were performed to characterize their specificities. Immunohistochemical staining was carried out on normal and cancerous breast tissue sections using the purified polyclonal Eag1-specific antibodies. The results indicate that the overexpression of Eag1 might be associated with an increased risk of progression to breast cancer (Grade 1 tissue=57.89%;Grade 2 tissue=92.59%;Grade 3 tissue=100%). These results also suggest that Eag1 gene is a putative growth-promoting gene that might be involved in breast tumorigenesis and development. Eag1 might further be represented as a potential target for some human diseases treatment.

Pipetting-driven microfluidic immunohistochemistry to facilitate enhanced immunoreaction and effective use of antibodies.

Immunohistochemistry (IHC), which has been used to detect antigens in cells of a tissue section using an immunoreaction between an antibody and an antigen, is a practical tool for identifying the type and stage of diseases in cancer diagnosis and scientific research. However, conventional IHC requires long, laborious process times and high costs. Although microfluidic IHC platforms have been developed to overcome these limitations, the application of microfluidic IHC in real-world environments is still limited due to the additional equipment needed to operate the microfluidic systems. In addition, continuous flow in a microfluidic channel leads to a waste of unbound antibodies. In this study, we demonstrate a novel and easy-to-use microfluidic IHC platform operated only using a manual pipette that is commonly available in research laboratories or hospitals. No other device such as a pump or a controller is required to operate our system. Bidirectional flows of the antibody solution in a microfluidic device are induced by repetitive manual pipetting which facilitates the enhanced antigen-antibody reaction and enables the effective use of a limited amount of antibody. When breast cancer cell and tissue sections are reacted with antibodies using our platform, pipetting for less than 2 min is sufficient to obtain immunostaining results without damaging the sample. The staining intensity by our method is similar to that of the sample stained for 1 h by a conventional batch process. We believe that this pipetting-based approach to the operation of a microfluidic system allows end users to use microfluidic IHC more conveniently and easily in real-world environments.

Dual-color chromogen In Situ hybridization (CISH) for the determination of Her2 status in breast cancer tissue sections

Background: A factor associated with breast cancer development is the mutation of the Her2/neu gene located on chromosome 17. Chromogen in situ hybridization (CISH) in which the color is generated with an enzymatic reaction is a promising alternative detection method to fluorescence in situ hybridization (FISH) which is nowadays used in clinical practice. The resulting color is stable and therefore usable for permanent storage, the use of a normal brightfield microscope is possible and the morphology of the sample can be evaluated more easily than with a fluorescence microscope. For the visualization of Her2/neu it is however important to also detect chromosome 17 and use it as a reference chromosome. The aim of the study is: to select the best substrates considering color production, background staining, reactivity and localization. Secondly to develop a dualcolor procedure visualizing Her2/neu and chromosome 17 to examine the possibility of using CISH as a standardized method for the detection of Her2 positive tumors and finally to compare these results with FISH. Methods: Single target CISH with eight substrates for the enzyme horseradish peroxidase (PO) and six substrates for alkaline phosphatase (AP) was performed on cervical cancer cell lines with centromere 1 (C1) and 1p36 as targets. The best substrates were subjectively selected with the use of a brightfield microscope. The procedure was expanded to a dual-color detection on cells and formalin-fixed, paraffin embedded (FFPE) breast cancer tissue sections with Her2/neu and centromere 17 (C17) as targets. In this procedure the best conjugate detection systems were worked out. Results: In the single target detection procedure the substrates DAB, Vina Green and Seramun Grun for PO and Ferangi Blue and Vulcan Fast Red for AP were found to give the best color reactions. The dual-color detection on tissue sections showed strong and good distinguishable color reactions when combining the substrates Vina Green for C17 and Vulcan Fast Red for Her2. Results of CISH and FISH were comparable when using immersion oil for the detection of the fluorescent signals. Discussion and Conclusion: Vina Green for C17 and Vulcan Fast Red for Her2/neu are the best substrates considering background staining, color distinguishability and localization and give comparable results to FISH. The study gives a further step in the direction of introducing CISH as a standardized method for the detection of Her2/neu amplification.

Electrospray deposition device used to precisely control the matrix crystal to improve the performance of MALDI MSI.

MALDI MSI has been recently applied as an innovative tool for detection of molecular distribution within a specific tissue. MALDI MSI requires deposition of an organic compound, known as matrix, on the tissue of interest to assist analyte desorption and ionization, in which the matrix crystal homogeneity and size greatly influence the imaging reproducibility and spatial resolution in MALDI MSI. In this work, a homemade electrospray deposition device was developed for deposition of matrix in MALDI MSI. The device could be used to achieve 1 μm homogeneous matrix crystals in MALDI MSI analysis. Moreover, it was found, for the first time, that the electrospray deposition device could be used to precisely control the matrix crystal size, and the imaging spatial resolution was increased greatly as the matrix crystals size becoming smaller. In addition, the easily-built electrospray deposition device was durable for acid, base or organic solvent, and even could be used for deposition of nanoparticles matrix, which made it unparalleled for MALDI MSI analysis. The feasibility of the electrospray deposition device was investigated by combination with MALDI FTICR MSI to analyze the distributions of lipids in mouse brain and liver cancer tissue section.

Intratumor Heterogeneity Affects Gene Expression Profile Test Prognostic Risk Stratification in Early Breast Cancer.

To examine the effect of intratumor heterogeneity (ITH) on detection of genes within gene expression panels (GEPs) and the subsequent ability to predict prognostic risk. Multiplexed barcoded RNA analysis was used to measure the expression of 141 genes from five GEPs (Oncotype Dx, MammaPrint, PAM50, EndoPredict, and Breast Cancer Index) in breast cancer tissue sections and tumor-rich cores from 71 estrogen receptor (ER)-positive node-negative tumors, on which clinical Oncotype Dx testing was previously performed. If the tumor had foci of high Ki67 (n = 26), low/negative progesterone receptor (PR; n = 13), or both (n = 5), additional cores were obtained. In total, 181 samples were processed. Oncotype Dx recurrence scores were calculated from NanoString nCounter gene expression data. Hierarchical clustering using all GEP genes showed that majority (61 of 71) of tumor samples clustered by patient, indicating greater interpatient heterogeneity (IPH) than ITH. We found a strikingly high correlation between Oncotype Dx recurrence scores obtained from whole sections versus tumor-rich cores (r = 0.94). However, high Ki67 and low PR cores had slightly higher but not statistically significant recurrence scores. For 18 of 71 (25%) patients, scores were divergent between sections and cores and crossed the boundaries for low, intermediate, and high risk. Our study indicates that in patients with highly heterogeneous tumors, GEP recurrence scores from a single core could under- or overestimate prognostic risk. Hence, it may be a useful strategy to assess multiple samples (both representative and atypical cores) to fully account for the ITH-driven variation in risk prediction. Clin Cancer Res; 22(21); 5362-9. ©2016 AACR.

The METINUS Plus method for nuclei quantification in tissue microarrays of breast cancer and axillary node tissue section

Abstract This paper presents the METINUS Plus (METhod of Immunohistochemical NUclei Segmentation Plus) method that has been developed for localization and quantification of the regulatory T cells’ nuclei. The proposed methodology performs color separation followed by the extraction and validation of objects. Objects categorized as clusters, based on the area and shape, are divided with locally applied watershed supported by color deconvolution. Objects are validated based on RGB, Lab color space, and color deconvolution data. The chosen validation criteria allow quantification and differentiation between the subpopulations of regulatory T cells and breast cancer cells, which express FOXP3. The evaluation is based on the quantity and quality of nuclei segmentation in comparison with the results of the manual counting, done by four pathologists on a set of 20 images. The results were analyzed in comparison with human evaluation using Kendall's tau-b correlation coefficient and the Wilcoxon signed-rank test. The main achievement of this study is a possibility to find criteria that allow differentiation between two types of immunopositive cells’ nuclei: regulatory T cells with homogeneous stained nuclei and all cells with FOXP3 expression; in both tumor and axillary node biopsies. A major advantage of computer image processing is the reproducibility of achieved results, thus minimizing human intervention, and providing traceable clinical information.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR.

The fibronectin III-1 domain activates a PI3-Kinase/Akt signaling pathway leading to αvβ5 integrin activation and TRAIL resistance in human lung cancer cells.

BackgroundFibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi-modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin’s unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII-1c) on TNF-related apoptosis inducing ligand (TRAIL) resistance.MethodsNCI-H460 non-small cell lung cancer cells were treated with FnIII-1c then assessed for TRAIL-induced apoptosis. Subsequent analysis of FnIII-1c-mediated signaling pathways was also completed. Human non-small cell lung cancer tissue sections were assessed for the expression of vitronectin by immunohistochemistry.ResultsFnIII-1c inhibited TRAIL-induced activation of caspase 8 and subsequent apoptosis in NCI-H460 lung cancer cells. FnIII-1c treatment was associated with the activation of the phosphatidylinositol-3-kinase/alpha serine/threonine kinase (PI3K/Akt) pathway and the αvβ5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non-small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor-stroma interface.ConclusionsUnfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvβ5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis.

Abstract 1840: Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays

Abstract Highly sensitive and quantitative whole transcriptome and surrogate transcriptome targeted sequencing TempO-Seq assays were used to profile gene expression from 1 mm sq focal areas of FFPE sections (5 μm thick) of normal and cancerous tissue within the same prostate. The whole transcriptome assay directly measures changes within the whole transcriptome. The surrogate assay targets fewer genes yet permits in silico extrapolation to identify changes across the whole transcriptome. Both approaches identified differentiating biomarkers and mechanistic pathways in prostate cancer, both corroborating what has been reported and adding new mechanistic insights which will be discussed. These new observations likely result from the sensitivity of the TempO-Seq assays and their robust performance measuring gene expression from focal areas of FFPE, which in turn permit improved isolation of histology. Benefits derived from the TempO-Seq platform which will be discussed are: 1) Insensitivity to RNA degradation (measurements from intact RNA RIN = 9.1 correlate to measurements from degraded RNA RIN = 3.0, R2 = 0.97). 2) Does not require RNA extraction. Measures total RNA from FFPE samples, both soluble RNA in the lysate and crosslinked insoluble RNA. 3) Simple capture-free ligation-based assay protocol, without 3’ or 5’ bias to the probes used to measure each gene. No requirement for poly-adenylation or capture of the RNA (typically required in ligation-based assays). 4) Single base specificity derived from the specificity of probe ligation. 5) Misligation rates &amp;lt;0.01% (unlike other ligation-based assays where misligation can be as great as 30%) enable no sample background controls to be run with typical counts of 0 for most genes and 1 to 3 counts for a few genes. 6) High signal-to-noise provides single cell sensitivity and ability to measure differential gene expression from small focal areas of tissue. 7) Excellent reproducibility (average ∼5% CV) between biological replicates across all expressed genes results in high significance of even small fold change differences. 8) Sequencing only the ligated probes complementary to a 50-base sequence of each gene eliminates the need for bioinformatics to analyze the sequencing data. Pooling of samples into a single sequencing run permits 42 or 865 surrogate samples and 8 or 153 whole transcriptome samples to be sequenced at the same time on the MiSeq or NextSeq, respectively, at an average of 250 counts/expressed gene. In combination, this results in low sequencing cost/sample without sacrifice of information or quality. 9) Simple, robust protocol requires only a PCR or qPCR instrument, and access to a sequencer. Citation Format: Milos Babic, Peter Shepard, Joanne Yeakley, Ruchir Shah, Deepak Mav, Raymond B. Nagle, Bruce Seligmann. Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays. [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 1840.

Abstract 3947: Atypical chemokine receptor ACKR3 expression is associated with aggressive behavior and poor prognosis in gastric cancer

Abstract Chemokine and their receptors are key mediators of normal physiology and a large number of pathologic conditions such as cancer, and this family of receptors is the emerging therapeutic target in the field of cancer treatment. ACKR3 is an atypical chemokine receptor first cloned from a dog cDNA library as the orphan receptor and was initially named Receptor Dog cDNA 1 (RDC1). Shortly after demonstrating that RDC1 binds with its ligand, stromal cell-derived factor-1α (SDF-1α) and interferon-inducible T-cell α chemoattractant (I-TAC), RDC1 was officially deorphanized. Accumulating evidence of recent studies have suggested that expression of ACKR3 is augmented in most of tumor cells as compared to their normal counterparts and is involved in cell proliferation, survival, migration, invasion during the initiation and progression of cancer. However, there is little information regarding their expression and clinical relevance in gastric cancer. The expression status of ACKR3 was investigated in 221 specimens of primary gastric cancer using immunohistochemistry. The correlation of ACKR3 expression with the clinicopathological features and survival outcomes was analyzed as well. Immunohistochemical staining of gastric cancer tissue sections revealed diverse cytoplasmic and membrane staining patterns for ACKR3. One hundred-fourteen cases (51.6%) showed low ACKR3 expression according to an arbitrary scoring system (grade 0-1; grade 0, n = 26; grade 1, n = 88), and one hundred-seven cases (48.4%) showed high expression (grade 2-3; grade 2, n = 58; grade 3, n = 49). There were no significant differences in age, gender, histology, tumor location, lymphatic and venous invasion among the two groups. However, high CXCR7 expression in cancer cells tended to be associated with proportion of tumor size greater than 5 cm (P = 0.055) and was significantly correlated with depth of tumor invasion (P &amp;lt; 0.001), lymph node metastasis (P = 0.042), and higher stage (P = 0.002). Furthermore, patients with high ACKR3 expression showed worse overall survival rate of 47% compared to 56% in patients with low ACKR3 expression. In conclusion, ACKR3 is differentially expressed in gastric cancer cells, and high expression of ACKR3 is associated with aggressive tumor behavior and poor survival in patients with gastric cancer, suggesting that ACKR3 plays an important role during gastric cancer progression. Citation Format: Nayoung Kim, Seung-Woo Baek, Hyewon Ryu, Yoon Seok Choi, Ik Chan Song, Hwan Jung Yun, Deog Yeon Jo, Samyong Kim, Hyo Jin Lee. Atypical chemokine receptor ACKR3 expression is associated with aggressive behavior and poor prognosis in gastric cancer. [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 3947.

Abstract 4944: Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay

Abstract The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors. Citation Format: Eric W. Martin, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay. [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 4944.

RBBP6: a potential biomarker of apoptosis induction in human cervical cancer cell lines.

Overexpression of RBBP6 in cancers of the colon, lung, and esophagus makes it a potential target in anticancer therapy. This is especially important because RBBP6 associates with the tumor suppressor gene p53, the inactivation of which has been linked to over 50% of all cancer types. However, the expression of RBBP6 in cancer and its interaction with p53 are yet to be understood in order to determine whether or not RBBP6 is cancer promoting and therefore a potential biomarker. In this study, we manipulated RBBP6 expression levels followed by treatment with either camptothecin or γ-aminobutyric acid in cervical cancer cells to induce apoptosis or cell cycle arrest. We began by staining human cervical cancer tissue sections with anti-RBBP6 monoclonal antibody to evaluate the extent of expression of RBBP6 in patients’ specimens. We followed on with silencing the overexpression of RBBP6 and treatment with anticancer agents to evaluate how the specimens respond to combinational therapy. Apoptosis induction was evaluated through confocal microscope, and flow cytometry using annexin V staining, and also by checking the mitochondrial and caspase-3/7 activity. Cell cycle arrest was evaluated using flow cytometry through staining with propidium iodide. RBBP6 was highly expressed in cervical cancer tissue sections that were in stage II or III of development. Silencing RBBP6 followed by treatment with γ-aminobutyric acid and camptothecin seems to sensitize cells to apoptosis induction rather than cell cycle arrest. Overexpression of RBBP6 seems to promote S-phase in cell cycle and cell proliferation. These results predict a proliferative role of RBBP6 in cancer progression rather than as a cancer-causing gene. Furthermore, sensitization of cells to camptothecin-induced apoptosis by RBBP6 targeting suggests a promising tool for halting cervical cancer progression.

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer.

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×108 M-1; ΔH = 4.32×104±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.

Abstract P1-02-01: Genomic analysis of single cells isolated by a pulse laser retrieval system

Abstract Background: Isolating tumor cells of interest and harvesting histologically pure samples is important for genomic studies. Laser capture microdissection (LCM) is an established method to obtain such purified cell populations for various applications including DNA, gene expression, and single cell analyses. However, LCM possesses problems such as limited optical resolution, cell fragmentation from dissection, and adherence of adjacent tissue to the cells which interrupts with single cell isolation from tissue sections. To overcome these obstacles, we developed a high-throughput pulse laser retrieval system which uses a wavelength that minimizes damage to the cellular content and is processed with a sacrificial layer that provides applicable optical resolution. The aim of this study was to evaluate the performance of the pulse laser retrieval system to provide appropriate samples for genomic analysis using breast cancer tissue. Methods: An indium tin oxide (ITO) coated glass slide was prepared using fresh frozen breast cancer tissue sections of 4㎛ thickness and stained by hematoxylin and eosin. The slide was mounted on the cell isolation machine and imaging was performed with a charge-coupled device camera using a 20× lens. Following identification of the target cells by a pathologist, nano-second pulsed laser (wavelength= 1064nm) was irradiated on the target. Isolated cells were collected in a polymerase chain reaction tube and whole genome amplification (WGA) was carried out using Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Amplified genomic DNA was fragmented and Illumina sequencing libraries were constructed. Sequencing was carried out to generate data with 0.1∼0.2× depth throughout the whole genome for each sample. Copy number variation (CNV) was analyzed by the Variable binning algorithm. Results: Whole genome amplification was performed using bulk tissue and 10 captured single cells from the same specimen. No difference in amplification coverage was observed between the two samples. A CNV analysis of captured single cells revealed similar CNV profiles with those in a matched bulk tumor. Whole exome sequencing (WES) of captured single cells yielded a variant frequency of 15% at a read depth of 15× and 50M base coverage, compared to 0% at 100× and 50M for WES using bulk tumor and 0.5% at 1200× and 100K for targeted sequencing using bulk tumor. Laser capture was performed for DCIS and stromal cells from the same slide. CNV analysis of the two samples showed minimal CNV in normal stromal cells in contrast to DCIS where multiple CNVs were observed. Conclusions: Newly developed pulse laser retrieval system is suitable for capturing single cells for genomic analysis of breast cancer. WGA, WES, and CNV analysis was successfully carried out using the captured single cells and showed no difference in profile compared to those performed with bulk tissue. This method may have the potential to replace LCM for certain applications such as single cell analyses. Citation Format: Lee H-B, Kim S, Lee K-M, Jung Y, Lee AC, Kim J, Bae S, Ryu HS, Yoo T-K, Moon H-G, Noh D-Y, Kwon S, Han W. Genomic analysis of single cells isolated by a pulse laser retrieval system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-02-01.