Bim Nuclear Translocation and Inactivation by Viral Interferon Regulatory Factor

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1–4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFβ receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy

Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90

P1-07-05: HER2 Status Resolution in FISH and IHC “Double Equivocal” Breast Carcinomas by Quantitative Real-Time PCR.

Abstract Background: Clinical testing for HER2 amplification/over-expression is performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) as outlined by the ASCO/CAP guidelines. Although these guidelines standardize testing and reporting, in a subset of patients, HER2 is equivocal by both IHC and FISH (“Double Equivocal”). These double equivocal patients represent a clinically problematic sub-group that currently lack standardized management guidelines. In this study, we utilize Quantitative Real-Time PCR (Q-RT-PCR) to resolve HER2 status in invasive breast cancer cases that could not be resolved via IHC and FISH testing. Material and Methods: FISH for HER2 was performed on 2259 invasive breast carcinomas from 1/2008 to 12/2010. In accordance with ASCO/CAP, all equivocal HER2 FISH cases were reflex tested by IHC. In double equivocal cases, RNA extraction was performed following macro-dissection using High Pure RNA Paraffin Kit (Roche Applied Biosciences, Indianapolis, IN). Q-RT-PCR was carried out using TaqMan® RNA-to-CT™ 1-Step Kit with primers and probes for HER2, B2M, and GAPDH (Applied Biosystems, Foster City, CA). Q-RT-PCR results were expressed as the relative quantification of HER2 vs. two control genes, all normalized against calibrator RNA from the MCF7 cell line. Cut off for Q-RT-PCR HER2 overexpression was set using ROC curve analysis (MedCalc, Belgium). Results: In our cohort of 2259 patients, 124 (5.5%) had an equivocal HER2 result by primary FISH testing. Reflex HER2 testing by IHC was unable to resolve the HER2 status in 35 (1.5%) patients. Detection of HER2 overexpression by Q-RT-PCR was validated using 50 FISH confirmed amplified and 50 non-amplified cases. Q-RT-PCR performed on these 2 control populations generated two non-overlapping populations and ROC curve analysis using a cut off value of 7.0 showed 100% sensitivity and specificity in detection of HER2 overexpression. Application of Q-RT-PCR in the double equivocal sub-group resulted in resolution of HER2 status in all cases, 8 HER2 positive (test value ranging from 7.12 - 15.37) and 14 HER2 negative (test value ranging from 1.05 - 6.92). Conclusion: Application of Q-RT-PCR for HER2 represents a viable approach to resolve HER2 status in cases that fail classification by both FISH and IHC. Q-RT-PCR combines the precision and high sensitivity of real-time PCR with the morphological specificity of histological evaluation and ultimately allows definitive HER2 classification at the time of initial diagnosis. This knowledge of HER2 status at the time of diagnosis allows for comprehensive neoadjuvant treatment although, additional studies correlating response to anti-HER2 therapy and HER2 status by Q-RT-PCR are warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-05.</jats:p

Abstract P1-02-04: Array based resolution of HER2 amplification status in breast carcinomas displaying apparent chromosome 17 aneusomy

Abstract Background: Molecular inversion probe (MIP) arrays offer high-quality copy number and genotype data with whole-genome coverage and high resolution of cancer related genes. The MIP array assay is specifically designed for use with Formalin Fixed Paraffin Embedded (FFPE) tissue due to its design which utilizes hybridization of short sequence inversion probes. In this study, we performed MIP array analysis on a cohort of breast carcinomas with putative chromosome 17 aneusomy identified by HER2/CEP17 dual probe FISH. We hypothesized that MIP array performed on cases with chromosome 17 aneusomy would allow HER2 amplification status resolution and provide additional clinically actionable data on genes with predictive and prognostic significance. Design: DNA was extracted from formalin fixed paraffin embedded tissue (Qiagen,Valencia, CA) from invasive breast carcinomas following macrodisection. Breast carcinomas utilized in this study segregated into three groups: 1) Aneusomic 2) Monosomic, and 3) Eusomic cases as determined by FISH utilizing a centromere 17 reference probe (PathVysion; Abbott Molecular). Matched patient normal lymph nodes were performed for a subset of cases from each group. MIP-array data was collected using the Affymetrix OncoScan™ array (Affymetrix, Santa Clara, CA). Analysis of array data was performed using Nexus software v6 (BioDiscovery, Inc., Hawthorne, CA). Result: Array-based detection of HER2 amplification in aneusomic breast carcinomas showed 100% concordance with single probe (HER2) FISH. However, based on genomic profiling, these cases showed great variability in regions of aneusomy along chromosome 17. In our aneusomic cohort, one region of chromosome 17 showed conserved eusomy status. This region was utilized for both array and FISH based ratio to resolve HER2 amplification status in 18 of 22 (82%) of cases. In addition to HER2 status resolution by MIP array, gains and losses in genes involved in modulating therapy were also identified, using this single assay. Conclusion: Use of MIP array-based genotyping as a single reflex assay in breast carcinomas with apparent chromosome 17 aneusomy resulted in HER2 amplification status resolution. In addition, use of this assay in our aneusomic cohort resulted in identification of a highly conserved region of chromosome 17 that is highly resistant to gains or losses in ploidy. The “D17S122” region was confirmed by FISH and use of a HER2/D17S122 ratio, allowed resolution of HER2 amplification status. In addition to HER2 status resolution and copy number data, MIP Array also provided comprehensive genomic coverage for analysis of non EGFR family genes potentially important for prognosis and therapy response prediction. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-04.</jats:p

Abstract P6-07-45: Molecular Morphology Based Genomic Signatures of Moderate Complexity Predict Pathologic Complete Response in <i>HER2</i> Molecular Breast Carcinoma Class Patients Treated with Trastuzumab-based Preoperative Therapy.

Abstract Background: Preoperative chemotherapy is an effective approach for “downstaging” some breast cancer patients, some of whom achieve a pathologic complete response (pCR), especially for non-luminal B HER2 positive molecular class (HNL). Determination of which HNL patients are more or not likely to achieve pCR would allow a more personalized, predictive approach to neoadjuvant management. Methods and Materials: Formalin-fixed biopsy specimens from 34 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color dual hapten bright field in situ hybridization (dual ISH) using repeat depleted locus specific probes (designation based on the name of a gene included within the targeted region) and a reference centromeric locus probe (CEN) on the same chromosome. Probe pairs included MET+CEN7, TOP2A+CEN17, PTEN+CEN10, and PIK3CA+CEN3. Only invasive carcinoma tumor cells were scored. In addition to assessing genomic gains and losses by the average number of gene loci or centromeres per cell and the ratio of gene loci to CEN, the percentage of cells with &amp;gt;2 gene or CEN signals (gene locus gain or CEN gain), &amp;lt;2 signals (gene locus loss or CEN loss), gene locus signals &amp;gt; CEN signals (gene locus/CEN gain), and gene locus signals &amp;lt; CEN signals (gene locus/CEN loss) were calculated for each parameter and cut point. The percentage of cells with either gene locus/CEN gain or loss was also evaluated. Sensitivities and specificities for detecting patients with pCR, based on either the high parameter being positive for pCR, and conversely based on the low parameter being positive for pCR, were calculated for each parameter and each cutoff. Receiver Operator Characteristics (ROC) curves were generated as sensitivity versus 1-specificity over all cutoffs tested, and Area Under the Curve (AUC) was calculated as one measure of a parameter's ability to distinguish patients with pCR from patients without pCR, with AUC = 1 being ideal and progressively lower values being less favorable. 2×2 contingency tables were evaluated at each cutoff to provide chi square probabilities as another measure of a parameter's ability to differentiate patients with pCR. Combinations of parameters were also evaluated by ROC and contingency table analyses. Results: MET/CEN7 gain or loss was predictive of pCR (AUC = 0.824, N=24) achieving 100% sensitivity and 69% specificity at a cutoff of 50% (X2 p &amp;lt; 0.00089). Combining this parameter with PIK3CA/CEN3 gain provided further improvement (AUC 0.937, N=24) with 89% sensitivity and 93% specificity achieved at respective cutoffs of 50 and 55%. Conclusion: Genomic signatures of moderate complexity generated from dual ISH evaluation of invasive breast carcinoma predict pathologic complete response in HER2 molecular class breast carcinoma patients treated with trastuzumab-based preoperative therapy. These findings require validation in additional patient cohorts. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-45.</jats:p

Abstract P1-02-05: Identification of HER-2/neu amplification using amplicon based benchtop next generation sequencing

Abstract Introduction Aplicon based next generation sequencing (NGS) assays have the potential to incorporate simultaneous assessment of somatic mutations and copy number variation into a single platform. Although not specifically designed for this purpose, we noted numerous examples of apparent HER-2/neu gene amplification in analyzing results from our mutation profiling 46-gene cancer NGS panel (Life Technologies, South San Francisco, CA). Material and Methods Existing amplicon coverage data from the TorrentSuite 2.0 (Life Technologies) pipeline was accessed and used to generate a HER-2/neu coverage proportion (ratio between all HER-2/neu amplicon reads and all other amplicon reads). This ratio was calculated for 170 unique cases of breast carcinoma. A total of 4 cases were eliminated from analysis due to indeterminate IHC/FISH results. Using a total of 166 cases, we performed ROC curve analysis to determine the sensitivity and specificity of NGS based HER-2/neu amplification compared to that of traditional IHC and FISH testing (MedCalc v8.0). Results The NGS based HER-2/neu coverage proportions in the 166 cases showed a clearly non-normal distribution with an outlier cluster of cases with an elevated NGS HER-2/neu ratio (6 cases with ratios &amp;gt;0.045). The distribution of NGS based HER-2/neu ratios for the entire population ranged from 0.01 to a maximum of 0.34. ROC curve analysis of NGS HER-2/neu proportions compared to IHC/FISH data showed a maximal sensitivity of 75.0% and specificity of 100% at an NGS ratio cut off of ≥0.045. In the 166 cases, 6 showed a HER-2/neu coverage proportion of &amp;gt; 0.045 (range 0.045-0.34). Utilizing the 0.045 NGS ratio cut off value, the population frequency of cases positive for HER2 expression was 3.4%. Of the 160 samples with NGS coverage proportion &amp;lt; 0.045, only two cases were positive by IHC/FISH testing. The correlation between NGS HER-2/neu coverage proportion and IHC/FISH testing was highly significant (Spearman rank correlation r = 0.67, p &amp;lt; 0.0001 CI: 0.58-0.75). Conclusions Even utilizing a limited somatic cancer panel that includes less than 200 amplicons in 46 genes, clinically significant HER-2/neu amplification can be readily identified. This is the first study to describe the functional utility of HER-2/neu amplification detection using a NGS based amplicon assay. This study shows that in addition to mutation analysis, amplification data can be simultaneously obtained, which has striking implications for potential clinical utility and HER-2/neu amplification detection outside of traditional IHC and HER2 testing modalities. Furthermore, utilizing this assay as a primary screening modality could limit the additional expense of multiple single gene testing. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-05.</jats:p

Abstract PD02-04: Automated Quantitative RNA In Situ Hybridization for Resolution of Equivocal and Heterogeneous ERBB2 (HER2) Status in Invasive Breast Carcinoma

Abstract Background: Breast carcinomas that demonstrate a heterogeneous ERBB2 (HER2) status or equivocal results by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) present diagnostic challenges for which there is neither a standard methodology to achieve resolution in the clinical laboratory nor a uniform approach to management. We assessed the feasibility of using a novel automated and quantitative HER2 mRNA bright field in situ hybridization (ISH) assay capable of single molecule detection to determine HER2 status in invasive breast carcinomas that demonstrated significant tumor heterogeneity or failed to be resolved by standard IHC and FISH algorithmic testing. Design: Formalin-fixed, paraffin-embedded (FFPE) breast carcinomas from a non-consecutive series of 163 patients were analyzed for HER2 mRNA using a fully automated bright field RNA ISH assay (RNAscope, Advanced Cell Diagnostics, Hayward, CA). Cases were assigned into either a training set (n = 34) or a validation set (n = 129) and analyzed by both Q-RT-PCR and RNAscope. automated image analysis was used to numerate the punctate signal dots per cell in RNAscope-stained slides. A HER2 mRNA score based on single-cell quantification by RNAscope was developed and correlated to HER2 FISH and HER2 mRNA Q-RT-PCR results. A simple cutoff value was derived using the training set and applied to the validation set. Results: Evaluable HER2 results were obtained for 154 cases (94.5%) by RNAscope and 163 cases (100%) by Q-RT-PCR. In the training set, both FISH/IHC positive and negative cases were definitively separated by both Q-RT-PCR and RNAscope. HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.77) and Q-RT-PCR (r = 0.81). Application of both methods to the validation set resulted in correct identification of 31/31 positive cases and 41/43 negative (overall concordance=97.3%) for both RNAscope and Q-RT-PCR. RNAscope showed a significant advantage over Q-RT-PCR in correctly identifying cases equivocal by FISH that were resolved by reflex IHC testing. RNAscope classified 7 of 26 (26.9%) FISH/IHC double equivocal cases as positives. In cases with HER2 protein heterogeneity, RNAscope showed a 100% concordance with FISH results, whereas Q-RT-PCR showed a 42.9% concordance. Conclusion: RNAscope analysis of HER2 mRNA is an effective means to resolve HER2 status in double equivocal cases and cases that demonstrate heterogeneity. Automation and image analysis-based quantification minimize analytical and post-analytical variability. Quantification of single RNA transcripts in situ at single-cell level demonstrates superiority over qRTPCR and great potential in predictive biomarker analysis. Further studies of larger cohorts correlating clinical response with HER2 mRNA expression in situ are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-04.</jats:p