Comparison of HercepTestTM 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 (TM) mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY (R) 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

Enhanced Antitumorigenic Effects in Glioblastoma on Double Targeting of Pleiotrophin and Its Receptor ALK

AbstractIn 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

Critical Role of Gα12 and Gα13 for Human Small Cell Lung Cancer Cell Proliferation <i>In vitro</i> and Tumor Growth <i>In vivo</i>

Abstract Purpose: In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. Although the contribution of the Gq/11-phospholipase C-β cascade to mitogenic effects in SCLC cells is well established, the relevance of G12/13 signaling is still elusive. In other tumor entities, G12/13 activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G12/13-dependent signaling in SCLC. Experimental Design: We used small hairpin RNA–mediated targeting of Gα12, Gα13, or both in H69 and H209 cells and analyzed the effects of Gα12 and/or Gα13 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 Gα12 and Gα13 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 Gα12 and Gα13 in SCLC and a marked p42/p44 activation upon Gα12/Gα13 knockdown. In a s.c. tumor xenograft mouse model, Gα12 or Gα13 downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, Gα12/Gα13 double knockdown completely abolished H69 tumorigenicity in mice. Conclusions: Gα12 and Gα13 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 G12/13 signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment. Clin Cancer Res; 16(5); 1402–15</jats:p

Abstract LB-244: Growth regulation in small cell lung cancer cells through G proteins: Analysis of Gq/11 and G12/13-dependent pathways

Abstract Background: The activation of G protein-coupled receptors by autocrine factors represents a principal growth regulating signal in small cell lung cancer (SCLC) cells, which relies on the concerted parallel activation of Gq/11- and G12/13-dependent signaling pathways. However, the molecular effectors of these pathways and the relative contributions of either signaling cascade to the cellular phenotype of SCLC cells are not clearly defined. Materials and Methods: In the present study we further characterised the molecular make-up and the phenotypic implications of Gq/11- and G12/13-dependent signaling in SCLC cells. For this purpose, we employed small hairpin RNA (shRNA)-mediated knockdown of G 12, G 13, or both, in the SCLC cell lines H69, H209, and H510. We analyzed the effects of G 12 and/or G 13 knockdown on mitogenic signaling cascades as well as on proliferation in vitro and tumor growth in vivo. Furthermore, we selectively modulated Gq/11-regulated effectors by employing shRNA-mediated gene knockdown, forced expression of mutant signaling molecules or pharmacological inhibitors. For comparison, a non-small cell lung cancer (A549) and a neuroendocrine cell line (PC-12) were also included. Results: In SCLC cell lines, lentiviral expression of shRNAs against G 12, G 13, or both, inhibited basal as well as bradykinine-promoted proliferation in vitro. In contrast, G 12 or G 13 knockdown did not exert antiproliferative effects in non-SCLC cells. The analysis of the activation status of all three major MAPK families revealed nonredundant functions of G 12 and G 13 in SCLC cells. In an s.c. SCLC xenograft mouse model, G 12 or G 13 downregulation resulted in reduced tumor growth due to decreased tumor cell proliferation. More strikingly, G 12/G 13 double knockdown completely abolished SCLC tumorigenicity in mice. Regarding Gq/11−-regulated signaling, we identified the non-receptor tyrosine kinase Pyk2 asa master switch which is able to promote either mitogenic or pro-apoptotic signaling. Pyk2 was indispensable to mediate neuropeptide/Ca2+-dependent activation of the Ras-ERK1/2 cascade, and the downregulation of Pyk2 severely inhibited proliferation of SCLC cells. On the other hand, overexpression of Pyk2 strongly induced apoptosis in SCLC, but not in PC12 cells. This pro-apoptotic effect of Pyk2 was independent of its central kinase domain, but dependent on an intact PPPKP717SRP motif responsible for activation of p130Cas-promoted signaling. Conclusions: The activation of G12/13 and Gq/11−signaling exerts a complex pattern of nonredundant effects in SCLC. In contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G12/13 signaling. The complete abolishment of in vivo tumor growth in our study suggests that RNAi-mediated double knockdown of G 12 and G 13, may provide a promising new avenue in SCLC treatment. Finally, Pyk2 represents a point of convergence of Gq/11-dependent pathways in SCLC cells and is able to promote either mitogenic or pro-apoptotic effects depending on the signaling context. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-244.</jats:p

Supplementary Data from Critical Role of Gα₁₂ and Gα₁₃ for Human Small Cell Lung Cancer Cell Proliferation <i>In vitro</i> and Tumor Growth <i>In vivo</i>

Supplementary Data from Critical Role of Gα12 and Gα13 for Human Small Cell Lung Cancer Cell Proliferation In vitro and Tumor Growth In vivo</i

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

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