Comparison of HercepTest™ mAb pharmDx (Dako Omnis, GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer: correlation with HER2 amplification and HER2 low status.

Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies.

FGFR3 mRNA overexpression defines a subset of oligometastatic colorectal cancers with worse prognosis.

OBJECTIVES: Metastatic colorectal cancer (CRC) remains a leading cause of cancer related deaths. Patients with oligometastatic liver disease represent a clinical subgroup with heterogeneous course. Until now, biomarkers to characterize outcome and therapeutic options have not been fully established. METHODS: We investigated the prevalence of FGFR alterations in a total of 140 primary colorectal tumors and 63 liver metastases of 55 oligometastatic CRC patients. FGF receptors (FGFR1-4) and their ligands (FGF3, 4 and 19) were analyzed for gene amplifications and rearrangements as well as for RNA overexpression in situ. Results were correlated with clinico-pathologic data and molecular subtypes. RESULTS: Primary tumors showed FGFR1 (6.3%) and FGF3,4,19 (2.2%) amplifications as well as FGFR1 (10.1%), FGFR2 (5.5%) and FGFR3 (16.2%) overexpression. In metastases, we observed FGFR1 amplifications (4.8%) as well as FGFR1 (8.5%) and FGFR3 (14.9%) overexpression. Neither FGFR2-4 amplifications nor gene rearrangements were observed. FGFR3 overexpression was significantly associated with shorter overall survival in metastases (mOS 19.9 vs. 47.4 months, HR=3.14, p=0.0152), but not in primary CRC (HR=1.01, p=0.985). Although rare, also FGFR1 amplification was indicative of worse outcome (mOS 12.6 vs. 47.4 months, HR=8.83, p=0.00111). CONCLUSIONS: We provide the so far most comprehensive analysis of FGFR alterations in primary and metastatic CRC. We describe FGFR3 overexpression in 15% of CRC patients with oligometastatic liver disease as a prognosticator for poor outcome. Recently FGFR3 overexpression has been shown to be a potential therapeutic target. Therefore, we suggest focusing on this subgroup in upcoming clinical trials with FGFR-targeted therapies.

Critical role of G(alpha)12 and G(alpha)13 for human small cell lung cancer cell proliferation in vitro and tumor growth in vivo.

PURPOSE: In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of G(q/11) and G(12/13) proteins. Although the contribution of the G(q/11)-phospholipase C-beta cascade to mitogenic effects in SCLC cells is well established, the relevance of G(12/13) signaling is still elusive. In other tumor entities, G(12/13) activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G(12/13)-dependent signaling in SCLC. EXPERIMENTAL DESIGN: We used small hairpin RNA-mediated targeting of G(alpha)(12), G(alpha)(13), or both in H69 and H209 cells and analyzed the effects of G(alpha)(12) and/or G(alpha)(13) knockdown on tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase (MAPK) activation. RESULTS: Lentiviral expression of small hairpin RNAs resulted in robust and specific G(alpha)(12) and G(alpha)(13) knockdown as well as markedly inhibited proliferation, colony formation, and bradykinin-promoted stimulation of cell growth. Analyzing the activation status of all three major MAPK families revealed nonredundant functions of G(alpha)(12) and G(alpha)(13) in SCLC and a marked p42/p44 activation upon G(alpha)(12)/G(alpha)(13) knockdown. In a s.c. tumor xenograft mouse model, G(alpha)(12) or G(alpha)(13) downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, G(alpha)(12)/G(alpha)(13) double knockdown completely abolished H69 tumorigenicity in mice. CONCLUSIONS: G(alpha)(12) and G(alpha)13) exert a complex pattern of nonredundant effects in SCLC, and in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G(12/13) signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment.

Enhanced antitumorigenic effects in glioblastoma on double targeting of pleiotrophin and its receptor ALK.

In adults, glioblastomas are the most lethal and most frequent malignant brain tumors, and the poor prognosis despite aggressive treatment indicates the need to establish novel targets for molecular intervention. The secreted growth factor pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) shows mitogenic, chemotactic, and transforming activity. Whereas PTN expression is tightly regulated during embryogenesis and is very limited in normal adult tissues, a marked PTN up-regulation is seen in tumors including glioblastomas. Likewise, the PTN receptor anaplastic lymphoma kinase (ALK) has been shown previously to be upregulated and functionally relevant in glioblastoma. In this study, we explore the antitumorigenic effects of the simultaneous ribozyme-mediated knockdown of both receptor and ligand. Various glioblastoma cell lines are analyzed for PTN and ALK expression. Beyond the individual efficacies of several specific ribozymes against PTN or ALK, respectively, antiproliferative and proapoptotic effects of a single gene targeting approach are strongly enhanced on double knockdown of both genes in vitro. More importantly, this results in the abolishment of tumor growth in an in vivo subcutaneous tumor xenograft model. Finally, the analysis of various downstream signaling pathways by antibody arrays reveals a distinct pattern of changes in the activation of signal transduction molecules on PTN/ALK double knockdown. Beyond the already known ones, it identifies additional pathways relevant for PTN/ALK signaling. We conclude that double targeting of PTN and ALK leads to enhanced antitumorigenic effects over single knockdown approaches, which offers novel therapeutic options owing to increased efficacy also after prolonged knockdown.

RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts.

RNA interference (RNAi) is a powerful strategy to inhibit gene expression through specific mRNA degradation mediated by small interfering RNAs (siRNAs). In vivo, however, the application of siRNAs is severely limited by their instability and poor delivery into target cells and target tissues. Glioblastomas are the most frequent and malignant brain tumors with, so far, limited treatment options. To develop novel and more efficacious therapies, advanced targeting strategies against glioblastoma multiforme (GBM)-relevant target genes must be established in vivo. Here we use RNAi-based targeting of the secreted growth factor pleiotrophin (PTN), employing a polyethylenimine (PEI)/siRNA complex strategy. We show that the complexation of chemically unmodified siRNAs with PEI leads to the formation of complexes that condense and completely cover siRNAs as determined by atomic force microscopy (AFM). On the efficient cellular delivery of these PEI/siRNA complexes, the PTN downregulation in U87 glioblastoma cells in vitro results in decreased proliferation and soft agar colony formation. More importantly, in vivo treatment of nude mice through systemic application (subcutaneous or intraperitoneal) of PEI-complexed PTN siRNAs leads to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth without a measurable induction of siRNA-mediated immunostimulation. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerts antitumoral effects. In conclusion, we present the PEI complexation of siRNAs as a universally applicable platform for RNAi in vitro and in vivo and establish, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM.

A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes.

RNA interference (RNAi) represents a powerful method for specific gene silencing. It is mediated through small double-stranded RNA molecules (small interfering RNAs, siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor that determines the success of RNAi is the ability to deliver intact siRNAs into target cells. Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density which function as transfection reagents based on their ability to compact DNA or RNA into complexes. This paper describes the application of lyophilized PEI/siRNA complexes based on a novel polyethylenimine. By fractionation of a commercially available 25-kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (PEI F25-LMW) with superior transfection efficacy and low toxicity in various cell lines is obtained. Complexes formed in 5% glucose, but not in 150 mM NaCl, can be lyophilized and reconstituted without loss of transfection efficacy. Furthermore, PEI F25-LMW is able to complex and fully protect siRNAs against nucleolytic degradation, and delivers siRNAs into cells where they display bioactivity. Upon lyophilization and reconstitution of PEI F25-LMW-based siRNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting of the growth factor VEGF. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF. In conclusion, siRNAs can be applied in lyophilized formulations, and lyophilized PEI F25-LMW-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.

Ribozyme-targeting reveals the rate-limiting role of pleiotrophin in glioblastoma.

Glioblastomas (GBMs) are the most frequent malignant brain tumors with very limited treatment options and nearly all GBM patients dying within 1 year. Pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) is a secreted growth factor that shows mitogenic, chemotactic and transforming activity. While PTN expression is tightly regulated during embryogenesis and very limited in normal adult tissues, a marked PTN upregulation is seen in tumors including glioblastomas. Targeting of the PTN receptors, ALK and RPTP-zeta, indicates a contribution of PTN-activated signaling pathways in glioblastomas. However, the relevance of PTN expression itself is unknown especially since, besides PTN, at least one more growth factor, midkine (MK), signals through ALK and is expressed in glioblastoma. Here we demonstrate the biologic relevance of PTN in 2 glioblastoma cell lines in vitro and in vivo. We show that stable ribozyme-targeting leads to a robust reduction of PTN mRNA and protein levels. This results in decreased cell proliferation, cell migration and soft agar colony formation in vitro. Comparing clonal ribozyme-transfected cells with different residual PTN levels, we establish a PTN gene-dose effect of glioblastoma cell proliferation. In a subcutaneous tumor xenograft mouse model, in vivo growth is markedly reduced upon PTN depletion, which is paralleled by decreased PTN serum levels. Furthermore, the immunohistochemical analysis of the tumors shows reduced angiogenesis in PTN-depleted tumors. We conclude that PTN is a rate-limiting growth factor in glioblastoma. Since PTN is overexpressed in glioblastomas but rarely found in normal tissue, PTN may represent an attractive therapeutic target.