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Background Cell spheroids and aggregates generated from three-dimensional (3D) cell culture methods are similar to in vivo tumors in terms of tissue morphology, biology, and gene expression, unlike cells grown in 2D cell cultures. Breast cancer heterogeneity is one of the main drug resistant mechanisms and needs to be overcome in order to increase the efficacy of drug activity in its treatments. Methods We performed a unique 3D cell culture and drug efficacy study with trastuzumab emtansine (Kadcyla®, T-DM1) across five breast cancer cell lines (BT-474, SK-BR-3, MDA-MB-361, MDA-MB-175, and MCF-7) that were previously investigated in 2D cell culture. We performed HER2 IHC staining, cell viability experiments, Gene-protein-assay (GPA), and T-DM1 internalization studies. Results We obtained significantly different results including higher IC50 for some of the cell lines. Our GPA showed some significant heterogeneous HER2 gene and protein expression in 3D cultured spheroids or aggregates. The fluorescent images also showed that a longer incubation time is needed for T-DM1 to be internalized effectively into 3D cultured spheroids or aggregates. Conclusion Our study demonstrated that the difference of T-DM1 drug activity in 3D spheroids or aggregates might be due to tumor heterogeneity and less efficient internalization of T-DM1 that is not seen using 2D cell culture models. Drug studies using 3D cell culture are expected to provide biologically relevant models for determining drug activity in tumor tissue in future drug response and resistance research.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.

Aberrant cellular signaling networks are implicated in major diseases including cancer, but are difficult to reliably quantitate, as many signaling proteins are expressed at low abundance and further reduced following specimen collection. MTIP is an integrated tissue-imaging platform that leverages bright, fluorescent reporters and sensitive spectral instrumentation, along with automated staining, image acquisition and non-parametric image analysis, to attain reproducible, multiplexed quantitation of signaling proteins in tissue. MTIP captured the phosphoactivity of six key PI3K/MAPK proteins (pAKTS473, pAKT308, pPRAS40, pS6, peIF4G and pERK1/2) at high precision (coefficient of variation, CV

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin-fixed paraffin-embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix-free laser desorption ionization time-of-flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.

Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures. Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiterTM-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent. Results: In 3D spheroids/aggregates, HER2 IHC staining and GPA assay showed for SK-BR-3 and BT-474 HER2 3+ expression and HER2/CEP17 of ≥ 2; MDA-MB-361 cells with HER2 2+ expression and HER2/CEP17 of ≥ 2.0; MDA-MB-175 cells with HER2 1+ expression and HER2/CEP17 < 2.0 and MCF-7 cells with HER2 0+(IHC staining only without HER2 GPA data). Some of the 3D spheroids/aggregates in MDA-MB-361 cells showed heterogeneous expression of HER2 protein. The IC50 values of 3D spheroids/aggregates for some cell lines were significantly higher than were demonstrated for cell lines grown in 2D cell cultures. The fold changes between 3D spheroids and 2D cells (72h T-DM1 treatment time) are: 4.2 for SK-BR-3; ≳ 10 for BT-474 and 22 for MDA-MB-361. Additionally, the fluorescent images showed that a longer incubation time was required for the T-DM1 drug (3 µg/ml) to be internalized effectively into BT-474 3D spheroids; for example, about 120h for 3D spheroids in comparison to about 36h in 2D cells. Interestingly, the 3D spheroids incubated for 120h with T-DM1 (470 µm) are smaller in size than 3D spheroids in the control group (600 µm) incubated for 120h without T-DM1 treatment. Conclusions: Drug efficacy studies performed on 3D cultured spheroids/aggregates are expected to be very important and biologically relevant for determining drug activity in tumor tissue. Our drug efficacy study using 3D cell culture demonstrated greater concentrations of T-DM1 and longer incubation times were required than for cells grown as 2D in some cell lines, likely due to less efficient internalization. Citation Format: Jean Z. Boyer, Gail Lewis Phillips, Hiro Nitta, Karl Garsha, Eric May, Brittany Admire, Robert Kraft, Megan Peccarelli, Andre Zamorano, Scott Gill, Eslie Dennis, Liz Vela, Penny Towne. Activity of trastuzumab emtansine (T-DM1) in 3D cell culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 29.

Loss of major histocompatibility complex class II (MHC II) expression is associated with poor patient outcome in diffuse large B-cell lymphoma (DLBCL). As MHC II molecules are lost with plasmacytic differentiation in normal cells, we asked whether MHC II loss in DLBCL is associated with an altered differentiation state. We used gene expression profiling, quantum dots, and immunohistochemistry to study the relationship between MHC II and plasma cell markers in DLBCL and plasmablastic lymphoma (PBL). Results demonstrate that MHC II(−) DLBCL immunophenotypically overlap with PBL and demonstrate an inverse correlation between MHC II and plasma cell markers MUM1, PRDM1/Blimp1, and XBP1s. In addition, MHC II expression is significantly higher in germinal center-DLBCL than activated B cell-DLBCL. A minor subset of cases with an unusual pattern of mislocalized punctate MHC II staining and intermediate levels of mRNA is also described. Finally, we show that PBL is negative for MHC II. The results imply a spectrum of MHC II expression that is more frequently diminished in tumors derived from B cells at the later stages of differentiation (with complete loss in PBL). Our observations provide a possible unifying concept that may contribute to the poor outcome reported in all MHC II(−) B-cell tumors.

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5′ fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.

Optical imaging techniques are being developed that promise to increase the information content related to specific molecular reporters. Such modalities do not produce contrast in the structural context of the surrounding tissue, making it difficult to reconcile molecular information with morphological context. We report a solution that enables visualization of the tissue morphology on formalin-fixed, paraffin embedded sections prepared for analytical biomarker imaging. Our approach combines modes of transmitted darkfield and fluorescence contrast and computer visualization to produce 2-component image data analogous to the classical hematoxylin and eosin histological stain. An interferometric hyperspectral image capture mode enables measurement of multiplexed biomarkers in annotated anatomic regions. The system enables practical correlative analysis of molecular changes within areas of anatomic pathology.

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin-fixed paraffin-embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix-free laser desorption ionization time-of-flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John WileySons, Ltd.

Cells of the moth immune system are derived from organs that loosely envelop the four wing imaginal discs. The immune response in these insects is believed to depend on the activities of two main classes of hemocytes: plasmatocytes and granular cells. The fates of cells that arise from these hematopoietic organs have been followed by immunolabeling with plasmatocyte-specific and granular-cell-specific antibodies. Cells within each hematopoietic organ differ in their coherence and in their expression of two plasmatocyte-specific surface proteins, integrin and neuroglian. Within an organ there is no overlap in the expression of these two surface proteins; neuroglian is found on the surfaces of the coherent cells while integrin is expressed on cells that are losing coherence, rounding up, and dispersing. A granular-cell-specific marker for the protein lacunin labels the basal lamina that delimits each organ but only a small number of granular cells that lie on or near the periphery of the hematopoietic organ. When organs are cultured in the absence of hemolymph, all cells derived from hematopoietic organs turn out to immunolabel with the plasmatocyte-specific antibody MS13. The circulating plasmatocytes derived from hematopoietic organs have higher ploidy levels than the granular cells and represent a separate lineage of hemocytes.

Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L1's complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies. Citation Format: Wenjun Zhang, Antony Hubbard, Adriana Racolta, Nick Cummins, Mehrnoush Khojasteh, Liping Zhang, Karl Garsha, Joerg Bredno, Dustin Harshman, Srabani Bhaumik, Tobin Jones, Marcin Kowanetz, Sanjeev Mariathasan, Ian McCaffery, Dustin Smith, J Andrew Williams, Lidija Pestic-Dragovich, Larry Morrison, Lei Tang. An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens. [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 5117.

It is important to have a working awareness of the many factors that can enhance, degrade or even distort the interpretation of quantitative data. Measurements in fluorescence microscopy may be discussed in the context of three major headings: (1) intensity, (2) spatial, and (3) temporal. The quantitative ability of instrumentation in each dimension is dependent on the performance characteristics of the instrument subsystems that contribute to the data gathering. In order for accurate and precise data to be recorded, not only must each subsystem perform well on its own merits, they must all be carefully orchestrated to work together in synergy. A number of basic considerations regarding the quantitative application of imaging instruments is outlined in the pages that follow; topics covered include detector technologies, illumination sources, optical limits, scan raster, specimen positioning and multi-channel acquisition.

The other chapters in this book give the reader an in-depth description of every important aspect of biological confocal microscopy that we could think of. This chapter is to provide the novice user of this instrument with a basic understanding of the practical information needed to use it effectively. Because the computer interfaces of the various commercial instruments vary greatly, this chapter will stress the important features of microscopical optics and the basics of sampling that are common to all instruments.

There is an increasing amount of interest in functionalized microstructural, microphotonic and microelectromechanical systems (MEMS) for use in biological applications. By scanning a tightly focused ultra-short pulsed laser beam inside a wide variety of commercially available polymer systems, the flexibility of the multiphoton microscope can be extended to include routine manufacturing of micro-devices with feature sizes well below the diffraction limit. Compared with lithography, two-photon polymerization has the unique ability to additively realize designs with high resolution in three dimensions; this permits the construction of cross-linked components and structures with hollow cavities. In light of the increasing availability of multiphoton imaging systems at research facilities, femtosecond laser manufacturing becomes particularly attractive in that the modality provides a readily accessible, rapid and high-accuracy 3-D processing capability to biological investigators interested in culture scaffolds and biomimetic tissue engineering, bio-MEMS, biomicrophotonics and microfluidics applications. This manuscript overviews recent efforts towards to enabling user accessible 3-D micro-manufacturing capabilities on a conventional proprietary-based imaging system. Software which permits the off-line design of microstructures and leverages the extensibility of proprietary LCSM image acquisition software to realize designs is introduced. The requirements for multiphoton photo-disruption (ablation) are in some ways analogous to those for multiphoton polymerization. Hence, “beam-steering” also facilitates precision photo-disruption of biological tissues with 3-D resolution, and applications involving tissue microdissection and intracellular microsurgery or three-dimensionally resolved fluorescence recovery after photobleaching (FRAP) studies can benefit from this work as well.

Multiphoton microscopy is becoming an increasingly popular modality of laser scanning microscopy for imaging living specimens. In order to improve the signal to noise ratio under the challenging imaging conditions typical of biomedical research, researchers may be forced to resort to measures which substantially increase the amount of energy to which specimens are exposed. Several mechanisms of damage to living cells compete to limit the power window for minimally invasive imaging in multiphoton microscopy; these mechanisms include heating due to linear absorption, phototoxicity related to multiphoton absorption, and optical breakdown due to multiphoton ionization. The relative contribution of each of these factors may change significantly depending on the specimen and imaging parameters. The present study investigates the dominant factors in limiting cell viability at moderate to high-energy fluence levels that may be necessary under non-ideal imaging conditions. The results of this study suggest that heating associated with optical breakdown is an important factor in limiting cell viability under difficult imaging conditions, and that this heating scales in a manner proportional to the energy fluence associated with a given set of scanning parameters. Also significant is the finding that cell viability does not appear to scale proportionally to pulse width in a manner consistent with 2-photon absorption at ultrashort pulse widths and moderate energy fluence levels. These results suggest that the use of a pre-compensation scheme to offset the positive group velocity dispersion due to laser scanning microscope optics is a practical means to increase the signal to noise ratio while minimally impacting cell damage thresholds at ultrafast (sub 200 femtosecond) pulse widths.

The use of fluorescence microscopy in the life sciences runs the gamut from basic photodocumentation to dynamic single-molecule fluorescence (SMF) studies. Recent advances in digital imaging technology are helping to expand the utility and popularity of many fluorescence microscopy techniques, including Forster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and fluorescence in situ hybridization (FISH). The newly created Microimaging Applications Group, which comprises Photometrics, Media Cybernetics, QImaging, Gatan, and MAG Biosystems, offers a broad range of innovative imaging solutions designed to enhance life science research capabilities.

Depending on the nature of the study and what sort of information one is trying to gather through the use of FRET, FLIM has some compelling advantages in certain situations, and can provide a quantitative evaluation of the donor, acceptor and FRET pair stoichiometry. It does require access to specialized equipment and software. Different approaches to FLIM data acquisition have different strengths and weaknesses. For dynamic studies requiring high time resolution, FLIM acquisition times can fall well short of ideal. If a yes/no answer to whether FRET is occurring is all that is required, then the polarization anisotropy of the acceptor can be used to determine FRET between fluorescent proteins (Rizzo and Piston, 2005). This is a relatively simple and robust method for confirming the presence/absence of FRET.

39 Background: ERG gene rearrangements lead to overexpression of the ERG oncogene in 50% of localized and metastatic prostate cancers. Recent studies have indicated that this gene fusion correlates with androgen sensitivity in prostate cancer patients and an association with increased DNA Repair activity (PARP1). The PTEN tumor suppressor has also been shown to regulate the AKT pathway, and related to increased PARP1 activity. We hypothesized that future PARP1 inhibitors and other targeted therapies for mCRPC will likely require the development of a companion diagnostic assay for measuring ERG rearrangement and PTEN deletion status in CTCs. Methods: We isolated CTCs utilizing the CellSearch system from mCRPC patients for enumeration. Following enumeration, the cells were transferred to glass slides and interrogated utilizing the automated Benchmark staining platform with multiplexed Quantum Dot FISH inclusive of probes for 5’ERG, 3’ERG, PTEN, and Cen 10. FISH signals were visualized utilizing spectral imaging. Results: Successful transfer of the CTCs from the CellSearch to a glass slide were achieved In model cell line systems, in cultured cells (LnCaP and VCaP) either directly or after spiking to PBL and five out of 6 times we were able to successfully demonstrate simultaneous single copy 5’ and 3’ ERG and PTEN/Cen 10 labeled probes. Studies are in progress to extend the clinical utility of this assay to isolated CTC from cases with metastatic prostate cancer. Conclusions: CTCs are likely to be utilized clinically for monitoring disease progression mCRPC patients as measures for therapeutic efficacy. As more therapeutics are approved for use in mCRPC patients, the ability for oncologists to molecularly characterize the type of CTCs driving tumor burden is likely to play a significant role in optimizing patient management by determining the type and the sequence of future targeted therapies. We have for the first time demonstrated a platform to molecularly characterize patient CTCs on an automated platform that is amenable to standard clinical use.

The identification of patients who will benefit from therapy is one of the more difficult questions in prostate cancer disease management. Approximately 60% of men with diagnosed prostate cancer will have an ETS rearranged tumor foci. The prostate cancer gene fusions identified thus far are characterized by the fusion of 5’ genomic regulatory elements (commonly androgen regulated), such as TMPRSS2, with the ETS family of transcription factors which include ERG, ETV1, ETV4, and ETV5. About 40-50% of prostate cancers have the TMPRSS2/ERG gene fusion which leads to the over expression of oncogenic transcription factors. The goal of this study was to develop a novel, integrated diagnostic testing method that accounts for tissue heterogeneity including multiple gene re-arrangements in single transformed nuclei. We selected a method that sequentially reflexes from an initial H&E to ERG immunohistochemistry (IHC), and finally to a quantum dot (Life Technologies) based FISH probes for the detection of multiple gene rearrangements in prostate cancer. To this end, probes specific to the ETS gene rearrangements including, 3’ and 5’ ERG, TMPRSS2, NDRG1, ETV1, and ETV4, were detected with up to four different quantum dot bioconjugates simultaneously in single nuclei. We have shown sensitive and specific detection of gene rearrangements with this testing method in the xenograft models, VCaP, H660, and LNCaP, as well as in samples from prostate needle biopsies and radical prostatectomies. In 6 of 88 cases, the ERG IHC was diagnostic of PIN (prostatic intraepithelial neoplasia) that was missed on initial examination of the H&E stain. Further, the four color quantum dot FISH assay inclusive of ERG gene rearrangements was confirmatory of ERG IHC reactivity. In addition, we also clearly demonstrated instances of multiple gene rearrangements in the 5’ and 3’ ends on TMPRSS2 and NDRG1 in the same cancer nuclei, but not in benign nuclei from the same tissue. The signal patterns associated with various genomic events assessed were: no rearrangement (normal), translocation through insertion, and translocation through deletion. In summary, we propose that the ERG antibody is likely to be a key component in a diagnostic PIN IHC cocktail, that follows the initial H&E, and may be reflexed to a multiplexed quantum dot FISH assay that incorporates the detection of prevalent gene rearrangements. This method may be useful as an aid in detecting clinically relevant diagnostic markers, since multiple gene rearrangements in the same cell may be identified early in prostate cancer progression. Studies are in progress to evaluate this testing strategy for prognostic value in assessing prostate cancer progression in selected prostate cancer and biopsy cohorts. At time of submission, assay is not approved for use in the US. 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 2218. doi:10.1158/1538-7445.AM2011-2218

TMPRSS2:ERG gene rearrangements are an early even found in 50% of prostate cancer patients. Identifcation of men who are at elevated risk of progression from neoplastic PIN (prostatic intraepithelial neoplasia) lesions to prostate carcinoma is an unaddressed, unmet medical need which may directly impact the practice of needle biopsy sampling and PSA screening. Previous studies have identified the presences of ERG rearrangements in ∼15% of High Grade PIN. In addition, ERG rearranged PIN lesions are highly associated with gene rearrangements in adjacent tumor foci on prostatectomy specimens. ERG gene rearrangements and protein over-expression in PIN may be associated with cancer in-situ, or may identify men who are at risk of more rapidly developing prostate carcinoma. The goal of this study was to identify the extent of ERG rearrangements in PIN lesions of the prostate. We selected to develop automated staining procedures to detect ERG over-expression by immunohistochemistry (IHC), as well as the specific gene rearrangements by a FISH assay. To this end, probes specific to the ERG gene rearrangements including 3’ and 5’ ERG, were detected with quantum dot (LifeTechnologies) bioconjugates. Fixatives commonly used in preservation of needle biopsies as well as fixation times were also evaluated. Specific FISH signals were deconvolved using an interferometer and spectral imaging software. We have shown sensitive and specific detection of gene rearrangements with this testing method in the xenograft models, VCaP, H660, and LNCaP, as well as in samples from prostate needle biopsies, and radical prostatectomies. In 6 of 88 prostate specimens, the ERG IHC was diagnostic of Low Grade PIN that was missed on initial examination of the H&E stain. Over-expression of ERG was not significantly associated with any specific type of gene rearrangement, including insertions or deletions in the 5’ region of the ERG gene. The ERG FISH test assessed rearrangements include: no rearrangement (normal), translocation through insertion, and translocation through deletion. In summary, the new anti-ERG immunohistochemistry assay coupled with the ERG FISH provide a highly sensitive and specific reflex test format that may have utility subsequent to the initial H&E, in detecting clinically relevant diagnostic markers in PIN lesions. Identification of gene rearrangements and ERG protein over-expression in Low Grade PIN may help define a new class of prostate disease. This test format may help identify men who should be considered for more aggressive monitoring, for example through re-biopsying and active PSA screening. Studies are in progress to evaluate this testing strategy for prognostic value in assessing prostate cancer progression associated with the incidence of PIN in selected prostate cancer and biopsy cohorts. At time of submission, assay is not approved for use in the US. 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 2227. doi:10.1158/1538-7445.AM2011-2227