Anthony E. Rizzardi

Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring

AbstractImmunohistochemical (IHC) assays performed on formalin-fixed paraffin-embedded (FFPE) tissue sections traditionally have been semi-quantified by pathologist visual scoring of staining. IHC is useful for validating biomarkers discovered through genomics methods as large clinical repositories of FFPE specimens support the construction of tissue microarrays (TMAs) for high throughput studies. Due to the ubiquitous availability of IHC techniques in clinical laboratories, validated IHC biomarkers may be translated readily into clinical use. However, the method of pathologist semi-quantification is costly, inherently subjective, and produces ordinal rather than continuous variable data. Computer-aided analysis of digitized whole slide images may overcome these limitations. Using TMAs representing 215 ovarian serous carcinoma specimens stained for S100A1, we assessed the degree to which data obtained using computer-aided methods correlated with data obtained by pathologist visual scoring. To evaluate computer-aided image classification, IHC staining within pathologist annotated and software-classified areas of carcinoma were compared for each case. Two metrics for IHC staining were used: the percentage of carcinoma with S100A1 staining (%Pos), and the product of the staining intensity (optical density [OD] of staining) multiplied by the percentage of carcinoma with S100A1 staining (OD*%Pos). A comparison of the IHC staining data obtained from manual annotations and software-derived annotations showed strong agreement, indicating that software efficiently classifies carcinomatous areas within IHC slide images. Comparisons of IHC intensity data derived using pixel analysis software versus pathologist visual scoring demonstrated high Spearman correlations of 0.88 for %Pos (p < 0.0001) and 0.90 for OD*%Pos (p < 0.0001). This study demonstrated that computer-aided methods to classify image areas of interest (e.g., carcinomatous areas of tissue specimens) and quantify IHC staining intensity within those areas can produce highly similar data to visual evaluation by a pathologist.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1649068103671302

Canonical Wnt/β-catenin Signaling Drives Human Schwann Cell Transformation, Progression, and Tumor Maintenance

Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of β-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.

Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.

CBP Loss Cooperates with PTEN Haploinsufficiency to Drive Prostate Cancer: Implications for Epigenetic Therapy

Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbp(pc-/-);Pten(pc+/-) mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp(-/-); Pten(+/-) and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27(KIP1), and p21(CIP1). Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbp(pc-/-);Pten(pc+/-) mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer.

Trp53 Haploinsufficiency Modifies EGFR-Driven Peripheral Nerve Sheath Tumorigenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.

The human immunodeficiency virus protease inhibitor ritonavir inhibits lung cancer cells, in part, by inhibition of survivin

Introduction: Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. Methods: RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. Results: Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 &mgr;M). Conclusions: Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Elevated hyaluronan and hyaluronan‐mediated motility receptor are associated with biochemical failure in patients with intermediate‐grade prostate tumors

The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan‐mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA‐related molecules with BF after prostatectomy in GS7 tumors.

Safe and effective sarcoma therapy through bispecific targeting of EGFR and uPAR

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo “ontarget” companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I–II study of 23 dogs with spontaneous, stage I–II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956–65. ©2017 AACR.

Expression of FGFR3 and FGFR4 and clinical risk factors associated with progression-free survival in synovial sarcoma

Although rare, synovial sarcoma (SS) is one of the most common soft tissue sarcomas affecting young adults. To investigate potential tumor markers related to synovial sarcoma prognosis, we carried out a single-institution retrospective analysis of 103 patients diagnosed with SS between 1980 and 2009. Clinical outcome data were obtained from medical records, and archived tissue samples were used to evaluate the relationship between progression-free survival (PFS) and several prognostic factors, including tumor expression of FGFR3 and FGFR4. No associations were found between PFS and gender, body mass index, tumor site, SS18-SSX translocation, or FGFR4 expression. As seen in previous studies, age at diagnosis (<35, 63% versus ≥35 years, 31% 10-year PFS; P = .033), histologic subtype (biphasic, 75% versus monophasic 34% 10-year PFS; P = .034), and tumor size (≤5 cm, 70% versus >5 cm, 22% 10-year PFS; P < .0001) were associated with PFS in SS patients. In addition, in a subset of patients with available archived tumor samples taken prior to chemotherapy or radiation (n = 34), higher FGFR3 expression was associated with improved PFS (P = .030). To the best of our knowledge, this is the largest study of SS to date to suggest a potential clinical role for FGFR3. While small numbers make this investigation somewhat exploratory, the findings merit future investigation on a larger scale.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Abstract Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.