Paraffin-embedded Cell Pellets Research Articles

Reference Samples to Compare Next-Generation Sequencing Test Performance for Oncology Therapeutics and Diagnostics.

Diversity of laboratory-developed tests (LDTs) using next-generation sequencing (NGS) raises concerns about their accuracy for selection of targeted therapies. A working group developed a pilot study of traceable reference samples to measure NGS LDT performance among a cohort of clinical laboratories. Human cell lines were engineered via CRISPR/Cas9 and prepared as formalin-fixed, paraffin-embedded cell pellets ("wet" samples) to assess the entire NGS test cycle. In silico mutagenized NGS sequence files ("dry" samples) were used to assess the bioinformatics component of the NGS test cycle. Single and multinucleotide variants (n = 36) of KRAS and NRAS were tested at 5% or 15% variant allele fraction to determine eligibility for therapy with the EGFR inhibitor panitumumab in the setting of metastatic colorectal cancer. Twenty-one (21/21) laboratories tested wet samples; 19 of 21 analyzed dry samples. Of the laboratories that tested both the wet and dry samples, 7 (37%) of 19 laboratories correctly reported all variants, 3 (16%) of 19 had fewer than five errors, and 9 (47%) of 19 had five or more errors. Most errors were false negatives. Genetically engineered cell lines and mutagenized sequence files are complementary reference samples for evaluating NGS test performance among clinical laboratories using LDTs. Variable accuracy in detection of genetic variants among some LDTs may identify different patient populations for targeted therapy.

Molecular Profiling for Supernatants and Matched Cell Pellets of Pleural Effusions in Non–Small-Cell Lung Cancer

Pleural effusion (PE) is commonly observed in advanced lung cancer patients. Cell-free total nucleic acid (cfTNA) isolated from cancer patients' plasma has allowed noninvasive tumor genome analyses; however, there are limited studies of detection and characterization of cfTNA in PE. Herein, we included 47 advanced non-small-cell lung cancer patients with PE, who had lung cancer driver mutations tested on tumor tissue specimens either at diagnosis or during disease progression. The supernatant and cell pellet of each PE were evaluated for molecular profiles in parallel on an Ion Torrent next-generation sequencing platform. Somatic mutations were detected in 89.1% supernatant cfTNA, but in only 54.3% of cell pellets. The overall concordance rate between supernatants and formalin-fixed, paraffin-embedded cell pellets at the mutation level was 53.3%. By contrast, 41.7% and 5.0% of somatic alterations were detected in supernatants and cell pellets, respectively. Furthermore, joint analysis of supernatants and cell pellets from PE showed a high concordance (88.3%) of variant detection with their respective tumor tissue specimens. Low-frequency T790M mutations in three cases (0.29%, 0.41%, and 1.56%) were detected in supernatants but not in the matched cell pellets or tumor tissues. In conclusion, pleural effusion-derived cfTNA can effectively be used in clinical practice for molecular analysis by next-generation sequencing, even in cases where corresponding cell pellets or tumor tissuesyield insufficient material.

Abstract P3-10-10: DYRK2 is a novel therapeutic target in ER negative breast cancer

Abstract Background Dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) belongs to a family of CMGC kinases that function as modulators of different downstream pathways that allow cells to cope with hypoxia, DNA damage and various stress signals. Additionally, DYRK2 has been implicated in various human cancers with both pro- and anti-tumour roles, which are probably cancer type- and cell type-dependent. Furthermore, studies show that DYRK2 is involved in epithelial-mesenchymal transition, hence suggesting a role in tumour metastasis. The current study investigates the prognostic role of DYRK2 in breast cancer and investigates its potential as a novel therapeutic target. Methods Immunohistochemistry was employed to investigate if nuclear expression of DYRK2 was associated with clinical outcome measures in a cohort of 715 patients. Expression was determined using the weighted histoscore method. Antibody specificity was confirmed in paraffin embedded cell pellets +/- DYRK2 silencing. Cell counts in parental and CRISPR-mediated DYRK2 knocked-out MDA-MB-468 and MDA-MB-231 cells (ER, PR, HER2, AR negative) were measured using Alamar Blue; NSGTMmice (n=8) were injected subcutaneously with MDA-MDB-231 with or without DYRK2 depletion to assess tumour growth in vivo. Results In a cohort of 715 patients, median follow-up was 160 months with 155 breast cancer deaths and 135 deaths due to other causes. The majority of patients were over 50 years of age (71%), had ductal carcinoma (88%), tumours <20mm in size (56%) and node negative disease (57%). 489 patients had ER positive disease, 226 had ER negative disease and of these 148 had TN (triple-negative) disease. DYRK2 expression was observed in the cell cytoplasm and nucleus and ranged from 3 to 200 weighted histoscore units (WHS) and ROC analysis was used to determine cut-offs, tumours with a cytoplasmic and nuclear WHS <145 were classified as low expression and tumours with a cytoplasmic and nuclear WHS >145 were classified as high expression. In the full cohort (p=0.087) and ER negative (p=0.066) cohort DYRK2 was not associated with cancer specific survival. However in TN disease high DYRK2 expression was associated with cancer specific survival (p=0.012, mean survival 145 months versus 107 months). This was potentiated in patients with ER, PR, HER2, AR negative disease (p=0.005, mean survival 166 months versus 100 months) and independent in multivariate analysis with age, histological tumour type, tumour size tumour grad, nodal status, ki67 index, chemotherapy, radiotherapy and recurrence (p=0.13, HR 3.920). Following this observation, patients with ER, AR negative disease were investigated and again high DYRK2 expression was associated with cancer specific survival (p=0.0003, mean survival 163 months versus 86 months) and was independent when combined in multivariate analysis (p=0.001, HR 4.154). To investigate if DYRK2 was a potential target in TN breast cancer, the effect of silencing DYRK2 was investigated. CRISPR-mediated DYRK2 depletion impeded cell proliferation in TN cell-lines and markedly reduced tumour burden in mouse MDA-MDB-231 xenografts (p<0.0001). Conclusions Our studies indicate that DYRK2 is indeed a potential therapeutic target for patients with TN breast cancer or ER, AR negative breast cancer. Citation Format: Edwards J, Baillie G, Quinn J, Monreno R, Banerjee S, Tomkinson N, MacKay S, De La Vega L. DYRK2 is a novel therapeutic target in ER negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-10.

P1.15-005 Relationship Between MYC Family Status and Sensitivity to Aurora Kinase Inhibitors in Neuroendocrine and Other Lung Cancer Cell Lines

Recently, it has been reported that MYC-driven SCLC cell lines exhibit synthetic lethality with Aurora kinase (AURK) inhibitors. The aims of this study are to evaluate sensitivities to neuroendocrine (NE) lung cancer cell lines with or without any MYC family overexpression and/or amplification using representative AURK inhibitors and to investigate the associations between drug sensitivities, MYC family status and NE differentiation. We screened a panel of 62 cell lines including 33 SCLC, 18 other NE, and 11 NSCLC, with various MYC family status for growth inhibition upon AURK inhibitors. MYC family copy number, gene expression, and protein status were examined by quantitative real-time PCR, microarray and Western blotting. Relative cell growth was analyzed by MTS assay, and selective AURK A and B inhibitors, MLN8237 (Alisertib) and AZD1152 (Barasertib), respectively, were used in this study. Cell lines with IC50 values <0.1 μM were defined as sensitive. Drug effects on cell cycle and morphology were determined by flow cytometry and examination of formalin-fixed paraffin embedded cell pellets. Of 31 NE cell lines with MYC family overexpression/amplification, 21 (68%) and 18 (58%) were sensitive to Alisertib and Barasertib, respectively. All 20 NE (and most NSCLC) cell lines lacking MYC family overexpression/amplification were resistant to AURK inhibitors. There was an excellent concordance (86%) between response to the two AURK inhibitors. Both MYC family overexpression and amplification were predictive of sensitivity; however, some overexpressing lines had normal copy number. Cell cycle analysis showed a mitotic arrest in some sensitive cell lines with treatments of these drugs. Representative sensitive cell lines showed cell ballooning and bizarre multinucleated polyploid cells, which indicated cell cycle arrest in G2/M. MYC family overexpression/amplification in NE lung cancer cell lines confers sensitivity to AURK inhibitors in approximately 70% of cases. Lack of MYC family overexpression/amplification was always associated with drug resistance. MYC family overexpression may occur in the absence of gene amplification, and, thus, overexpression is a better predictor of sensitivity than amplification. We are currently investigating the mechanism of resistance in overexpressing NE cell lines and determining whether NE differentiation plays a role in drug sensitivity.

Abstract P1-03-10: HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles

Abstract Background: The human epidermal growth factor receptor-2 (HER2) is a member of a family of transmembrane tyrosine kinase receptors that play an important role in regulated normal cell growth and differentiation. The over-expression of HER2 in a subset of 15-20% of invasive breast cancers has an important bearing on prognosis, as HER2-positive tumors are associated with an aggressive clinical course and poor outcome. Targeting HER2-overexpression has been shown to be a remarkably effective therapeutic modality; however testing of tumor samples to assess the HER2 status of the patient's breast cancer is required. Clinical assays to assess the HER2 status in patients being considered for targeted therapy include immunohistochemistry (IHC), which detects protein over-expression, or fluorescence in situ hybridization (FISH), which detects gene amplification. Both the IHC and FISH methodologies have limitations. Given that the target of the currently approved drugs is the receptor protein, novel detections systems that could more accurately and quantitatively detect HER2 protein in clinical samples over a broad dynamic range would be advantageous and may be clinically helpful. Material and Methods: A novel detection technology using streptavidin-coated Phosphor Integrated Dot fluorescent nanoparticles (PID) has been developed that can be visualized by fluorescence microscopy and used for quantitative immunofluorescence detection of protein in clinical samples using computer assisted image analysis. In the current study, PID- nanoparticles were used to analyze HER2 protein expression in breast cancer cell lines and 120 well characterized breast cancer samples. These results have been compared with HER2 IHC and HER2 FISH analysis. Results: The expression levels of HER2 protein from 8 breast cancer cell lines was evaluated by antibody-binding capacity with FACS analysis. Formalin fixed paraffin embedded cell pellets for these cell lines were prepared and used for quantitative HER2 analysis by PID. The PID score/cell for each of these cell lines showed a strong linear correlation with antibody-binding capacity sites/cell by FACS analysis (R2 = 0.94). For the 120 breast cancer samples, PID score/cell was measured and compared against HER2 IHC membrane intensity measure by image analysis (Aperio) and HER2 FISH results. The HER2 PID score/cell showed a correlation coefficient of R2=0.72 versus the average HER2 copy number per cell by FISH, compared with a correlation coefficient of R2=0.41 for HER2 IHC membrane intensity measured by Aperio. For the HER2/CEP17 ratio, the correlation coefficient for the PID score/cell was R2=0.79 compared with a correlation coefficient of R2=0.32 for the HER2 IHC membrane intensity. Conclusions: PID-nanoparticles demonstrate great potential for the quantitative measurement of protein of clinical interest in routine clinical samples with morphologic confirmation of the tissue being studied. Further studies looking for PID-score thresholds for HER2 gene amplification and correlations with clinical outcome data are warranted and ongoing. Citation Format: Hicks DG, Goda H, Zhai H, Okada H, McMahon L, Sullivan N, Tang P, Nakano Y. HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-10.

Cell Line Macroarray: An Alternative High-Throughput Platform to Analyze hiPSC Lines.

In the past decade, tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly, enormous amount of data can be obtained from the cell line macroarray (CLMA) technology, which developed from the TMA using formalin-fixed, paraffin-embedded cell pellets. Here, we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones, which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here, we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer, TissueQuest, as a reliable tool to quickly select the best clones, based upon the level of expression of multiple pluripotent biomarkers.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours

BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.

Abstract 742: Comparison of commercially available phosphorylated ERK antibodies for immunohistochemical biomarker analysis

Abstract Immunohistochemical (IHC) analysis of ERK phosphorylation is commonly performed on pre- and post-treatment cancer biopsies as part of pharmacodynamic biomarker assessments for targeted oncology therapeutics. The current study was performed to assess the specificity and sensitivity of four commercially-available pERK antibodies for IHC analysis, which included three monoclonal antibodies (clones 20G11, MAPK-YT, D13.14.4E) and one polyclonal antibody. To facilitate this analysis, the HEPG2 liver cancer cell line was grown in the presence or absence of inhibitors of the RAF/MEK/ERK pathway (U0126 or sorafenib). Cell lysates were prepared for analysis via Western blotting and formalin-fixed, paraffin-embedded cell pellets were prepared for analysis via IHC. All antibodies produced bands at 44 and 42 kDa, the expected molecular weights of ERK1/2, when used for Western analysis of untreated cells and demonstrated a reduction or ablation of these bands following treatment of cells with inhibitors of the RAF/MEK/ERK pathway. When examined via IHC, the monoclonals demonstrated nuclear and cytoplasmic staining in HEPG2 cells with increased sensitivity relative to the polyclonal antibody. The monoclonals also demonstrated greatly reduced staining in HEPG2 cells treated with inhibitors of the RAF/MEK/ERK pathway. These antibodies were further characterized for reactivity in human tumor specimens representing breast cancer, colon cancer, lung cancer, melanoma, and ovarian cancer. Melanoma tissues were stained with a red chromogen; all others were stained with DAB. Differences in the frequency of expression, percentage of expression, and protein localization were identified between the three monoclonals. In summary, this work has identified commercially-available antibodies that appear to be of suitable specificity and sensitivity for use in IHC assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 742. doi:1538-7445.AM2012-742

Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.

Abstract 2235: FRET assay to assess EGFR/HER2 dimerization in cancer cell lines

Abstract Expression level of the HER family is unreliable as a predictive marker for the outcome of targeted therapies in cancer. Only a third of HER2 positive breast cancer patients respond to trastuzumab monotherapy. Thus there is significant need for the development of new biomarkers and the role of dimerization may prove a more effective predictor than expression itself. It has been shown that specific HER dimerization pairs are important. However, there has been no protocol proposed which can accurately assess dimerization to facilitate the development of an assay viable for use in a clinical setting. Previous attempts have either been based upon transfection of proteins whose interactions can be monitored which would not seem to be directly applicable to in-situ monitoring or do not explicitly separate true dimerization from mere colocalization. Our approach also allows intracellular localization to be visualized, this is significant as nuclear translocation of EGFR can lead to a number of signaling events. Utilizing measurement of Foerster Resonance Energy Transfer (FRET) to assess distances of the order 7 nm between receptors we can quantify dimerization. Our secondary conjugation technique uses anti-mouse and anti-rabbit primary antibodies and species specific dye-conjugated secondary antibodies, with Alexa488 as the donor and Alexa546 as the acceptor dye. An increase in dimerization results in increased occurrence of FRET which can be measured as a reduction in donor fluorescence lifetime. We have validated our assay for dimerization of EGFR and HER2 in 3 cell lines, BT474, SKBr3 and A431, and furthermore in paraffin embedded cell pellets. Comparison of preparations with donor antibodies alone with those containing donor and acceptor we demonstrate basal EGFR/HER2 dimerization levels are present (p&amp;lt;0.025). This was enhanced by ligand EGF, producing further decrease in lifetime (p&amp;lt;0.025). Whereas tyrosine kinase inhibitor gefitinib and HER3/HER4 ligand heregulin, which decreases EGFR/HER2 dimerization, showed an increase in lifetime. These results are consistent with documented changes seen using co-immunoprecipitation experiments. We then applied the assay to monitor response to targeted therapies, the HER2 specific monoclonal antibodies, trastuzumab and pertuzumab. Trastuzumab is seen to decrease lifetime while dimerization inhibitor pertuzumab produces an increase. This is consistent with findings using biochemical methods. Finally, we applied and validated our assay in paraffin embedded cell pellets. These data demonstrate the robustness of our assay and the potential clinical application to paraffin-embedded tissue microarrays in order to assess the contribution of HER signaling in-situ. The assay represents a proof of principle, from here this same technique could be applied to other dimerization pairs. This will allow the prognostic potential of dimerization to be assessed and used to inform treatment regimes. 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 2235. doi:10.1158/1538-7445.AM2011-2235

Abstract 304: Comparison of commercially available phospho-AKT [Ser473] antibodies for immunohistochemical biomarker analysis

Abstract Immunohistochemical (IHC) analysis of AKT phosphorylation on Ser473 is commonly performed on pre- and post-treatment cancer biopsies as part of pharmacodynamic biomarker assessment for targeted oncology therapeutics. The current study was performed to assess the specificity and sensitivity of four pAKT Ser473 monoclonal antibodies for IHC analysis (clones D9E, 736E11, 587F11 and 14-5). To facilitate this analysis, cancer cell lines with constitutive activation of the PI3K pathway due to mutations in PIK3CA (MCF-7 and MKN-1) or PTEN (MDA-MB-468) were grown in the presence or absence of inhibitors of the PI3K pathway (wortmannin and LY294002). Cell lysates were prepared for analysis via Western blotting and formalin-fixed, paraffin-embedded cell pellets were prepared for analysis via IHC. All antibodies produced a band at 65 kDa, the expected molecular weight for pAKT, when used for Western blot analysis of untreated cell lines and demonstrated a reduction or ablation of this band following treatment of the cell lines with inhibitors of the PI3K pathway. When examined via IHC, antibodies 736E11 and 587F11 demonstrated nuclear and cytoplasmic staining in MCF-7 and MKN-1 cells and membrane staining in MDA-MB-468 cells. These antibodies demonstrated a reduction of staining in all three cell lines treated with PI3K inhibitors, but the reduction was modest in MCF-7 and MKN-1 cells. Clone 587F11 was the least sensitive for IHC. Staining with antibodies 14-5 and D9E was predominantly cytoplasmic or membrane associated, while clone14-5 produced a more punctate appearance. Strong staining was achieved with clone 14-5 and prominent reduction or ablation of staining was observed in all three cell lines treated with PI3K pathway inhibitors. Staining with clone D9E was a little less sensitive than with clone 14-5, but significant reduction or ablation of staining in cell lines treated with PI3K inhibitors was also observed. In summary, clones 14-5 and D9E appear to demonstrate adequate specificity and sensitivity for use in IHC assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 304.

Abstract A31: Development of an immunohistochemistry-based pharmacodynamic biomarker program for phase I trials of MLN4924, an inhibitor of the NEDD8 activating enzyme

Abstract The novel small molecule MLN4924 is an inhibitor of the NEDD8 activating enzyme (NAE) and is currently in Phase I trials. As part of the ubiquitin-proteasome system, NAE is required for activity of cullin ring ligases (CRLs), a category of ubiquitin E3 ligases. When NAE is inhibited, levels of CRL substrates increase due to impaired ubiquitination and proteasomal degradation; CRL substrates can therefore serve as pharmacodynamic (PD) biomarkers of NAE pathway inhibition. We describe the development of Immunohistochemistry (IHC) assays for two CRL substrates: NRF2 (a transcription factor involved in the oxidative stress response) and CDT1 (a protein that regulates DNA replication). CDT1 and NRF2 IHC assays were initially evaluated in PD studies of xenograft tumors grown in immunocompromised mice and showed dose-dependent regulation. The IHC assays were then adapted for use in clinical samples, including skin and tumor biopsies from patients in Phase I trials of MLN4924. Sample quality and morphology were assessed by pathologist review of hematoxylin and eosin pathology stain. A set of formalin-fixed, paraffin-embedded cell pellets was evaluated with each IHC run to detect technical variability and establish pass/fail criteria for each experimental IHC run. The QC samples were generated from the HCT116 cell line treated with various concentrations of MLN4924. The QC controls were assayed multiple times to generate an expected range as a percent positive area above a threshold for each concentration of MLN4924. The ranges were then applied to each experimental run to pass or fail the experiment. In cases of assay failure, the clinical samples were re-stained. Skin and melanoma tumors are inherently challenging for IHC analysis due to the population variability of melanin pigment, which can be indistinguishable from brown DAB chromagen. Adaption of the assays to a blue chromagen (NBT/BCIP) enabled a contrast between the chromagen stain and endogenous pigments. A region of interest (ROI) for skin was manually drawn to select the epidermal area. Semi-automated image analysis was used to measure percent positive area for CDT1 and NRF2 within the total epidermal region. To determine the baseline levels for CDT1 and NRF2 signal, 10 benign skin samples were studied. Staining of six sections at 10-micron increments was performed on each sample to investigate both the inter- and intra-patient variability. Overall, the percent positive area of both substrates was below 1% in benign skin and variation from section to section was minimal. To survey the baseline levels of NRF2 and CDT1 across a variety of tumor types, we evaluated archival tumors of various types including melanoma, head &amp; neck squamous cell carcinoma, prostate, lung, and breast cancer. A manual ROI was identified by pathologist review to select for tumor area, and semi-automated image analysis was used to measure the percent positive area for NRF2 and CDT1 within the total tumor area. Unlike skin, the tumor samples showed variable levels of NRF2 and CDT1. However, most samples showed a percent positive area less than 40%, indicating the feasibility of detecting a PD effect by comparing pre- and post-dose tumor biopsies. In summary, reliable IHC PD biomarker assays have been established for the NAE inhibitor MLN4924. The assays are currently being used to measure PD effects of NAE inhibition in skin and solid tumors from Phase I trials of MLN4924. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A31

Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

BackgroundChlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated.MethodsWe validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-γ) induced model of chlamydial persistence.ResultsIn the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-γ treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-γ treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-γ persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC.ConclusionTMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

Expression of the MRP and MDRI Multidrug Resistance Gene Products in 160 Untreated Human Carcinomas Studied by Immunohistochemical Methods in Formalin-Paraffin Sections

An immunohistochemical method was developed and applied to detect multidrug resistance related protein (MRP) in sections of formalin-fixed, paraffin-embedded human tissues. Monoclonal antibodies MRPm6, MRPrl, and QCRL-1 were used on sections of paraffin-embedded cell pellets of known MRP expression (HL60, HT1080, HeLa). None of the antibodies succeeded without pretreatment, but microwave epitope retrieval with 6M urea resulted in excellent specific staining with MRPm6. Moderate or weak staining was seen with MRPrl and QCRL-1. Various carcinomas were tested for MRP with MRPm6, and for MDRl P-glycoprotein (Pgp) expression with MAb JSB-1, by our previously described method (Am J Pathol 144:227-236, 1994) to investigate the possible coexpression of multidrug resistance markers in the same solid tumor. The incidence of positive immunoreactions/case with MRPm6 and JSB-1 respectively, in various human cancers was as follows: lung, 15 and two of 26; esophagus, eight and two of 15; head and neck, nine and one of 27; colorectal, 13 and 11 of 20; breast, 25 and 23 of 55; bladder, 0 and 0 of seven; ovarian, 0 and 0 of 10 cases. The overall incidence of MRP expression in these tumors was higher 70/160 (44%) than that of Pgp 41/160 (26%). Among Pgp-negative tumors 38% proved to be MRP positive. Coexpression of MRP and Pgp was found in 25/160 cases (16%), which is statistically significant (p=.0 17). A relatively higher incidence of strong MRP-positive staining was found in lung and esophageal cancers (4/26 and 5/15); otherwise staining was weak to moderate. No detectable MRP was found in stromal cells and in normal organs. Thus, MRPm6 by this method allows detection of MRP overexpression versus normal cells in paraffin sections of archived surgical specimens for investigation of the clinical significance of MRP.

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Composition of human islet cell preparations for transplantation.

To study the cellular composition of human islet cell isolates for transplantation, formalin-fixed and paraffin-embedded cell pellets were stained by the immunoperoxidase method with a panel of antibodies characterising endocrine, epithelial, soft tissue and haematolymphoid components. Immediately after separation, the isolates contained 30-80% islet cells, differing mainly in the content of islet and acinar cells, whereas the soft tissue, ductal/ductular and haematolymphoid elements comprised a relatively constant 10-20%. After 1 week in culture the islet cell content of less highly purified isolates (30-40% islets) dropped dramatically to 5%. The highly purified isolates (70-80% islets) showed only a minimal change in cellular composition; however, approximately two-thirds of islet cells were degranulated and did not stain for insulin. Haematolymphoid components were still present in all cultured isolates. We conclude that primarily mechanical purification methods and short-term culture are not sufficient to eliminate highly immunogenic cells. In addition, short-term culture is deleterious to the isolate if a significant number of acinar cells is still present after enrichment.

Comparison of 35S and biotin as labels for in situ hybridization: use of an HPV model system.

Colorimetric in situ hybridization is a method of potential importance in diagnosis and research. The largest criticism of the method has been a perceived loss of sensitivity compared with autoradiographic techniques. Our more positive experience with automation of colorimetric in situ hybridization led us to undertake a direct comparison of the sensitivity of 35S- and biotin-labeled probes. Serial sections of formalin-fixed, paraffin-embedded cell pellets from four human cervical carcinoma cell lines with known copies of HPV (CaSki, 400-600 copies HPV 16; HeLa, 10-50 copies HPV 18; SiHa, 1-2 copies HPV 16; HTB31, no known copies HPV) were hybridized with protocols optimized for autoradiographic or colorimetric detection. Both methods gave comparable results, with differences in each technique seen at the limits of sensitivity. The 1-2 copies of HPV 16 per SiHa cell can be detected with both methods; however, grain counting is required for interpretation of the autoradiographic result. This degree of sensitivity for colorimetric in situ hybridization in formalin-fixed, paraffin-embedded material is achieved through careful optimization of probe size and labeling, adequate tissue digestion, and removal of background. Autoradiography may be preferred in situations where quantitation is required, but colorimetric detection retains the advantages of speed, potential for automation, and improved localization of signal with comparable sensitivity.