Zhang, Qing Hua

9091 Background: Biomarker testing, including for rearrangement during transfection (RET), is recommended and important in selecting efficacious targeted therapy for patients with advanced non-small cell lung cancer (aNSCLC). This retrospective cohort study characterized RET testing and treatment patterns among aNSCLC patients in clinical practice. Methods: We used the nationwide Flatiron Health electronic health record-derived de-identified database to select patients diagnosed with aNSCLC (stage ≥IIIB) from 2017-2022. We measured rates of RET testing (test result ≤90 days after advanced diagnosis). We used logistic regression to identify baseline characteristics predictive of timely testing. Finally, we described treatment patterns for patients with RET+ aNSCLC before and after the US approval of RET inhibitors. Results: Among 26,329 aNSCLC patients, rates of RET testing ≤90 days after advanced diagnosis increased from 25% (2017 Q1) to 73% (2022 Q1). Patients were less likely to receive RET testing within 90 days if they were Black (multivariate odds ratio [OR] 0.8) or Hispanic (OR 0.8) compared to White, or had Medicare (OR 0.9; all p<0.001) compared to commercial insurance. Patients were more likely to receive RET testing within 90 days if they were in the highest socioeconomic quintile (OR 1.4), had de novo disease (OR 2.1), or no history of smoking (OR 1.3; all p<0.001). Between 2017 and 2022, there were increases in the use of next-generation sequencing (66% to 89%) and blood samples for biomarker testing (25% to 49%) and a decrease in mean days from diagnosis to test result (33 to 28) for the first RET test among those who had testing within 90 days. Among all patients with aNSCLC tested within 90 days (n=12,275), 73 were RET+ and received 1L treatment ≤90 days after advanced diagnosis. The most common 1L treatments before (n=35) and after (n=38) RET inhibitor approval were chemotherapy (40%) and RET inhibitor (42%), respectively (Table). Conclusions: RET testing has increased over time, but certain patient subgroups are less likely to receive timely RET testing and therefore may not be optimally treated. Among patients with RET+ aNSCLC identified after RET inhibitor approval, only 42% were treated with a RET inhibitor in the 1L setting. These results suggest both disparities in access to RET testing and unmet medical need among patients with RET+ aNSCLC. [Table: see text]

Abstract Background: High Grade Serous Ovarian Cancer (HGSOC) is a highly aggressive form of ovarian cancer accounting for up to 70% of all cases. The Cancer Genome Atlas (TCGA) has further defined 4 molecular subtypes within HGSOC: differentiated (DIF), immunoreactive (IMM), mesenchymal (MES), and proliferative (PRO). The MES subtype is associated with the worst prognosis and the least likely to be completely resectable when compared with other subtypes. A recent study suggested the existence of a 5-th, so-called anti-mesenchymal (ANM), subtype which is characterized by down-regulation of genes typically upregulated in the MES subtype. Importantly, previous molecular subtype studies have been performed using tumor samples consisting of both cancer cells and stroma. However, the origin of MES biomarkers in OC is still largely unknown and several studies suggest that many of the MES biomarkers could arise from cancer-associated stroma rather than cancer cells. Methods: Specimens from 20 patients (MES subtype, n=15; ANM subtype, n=5) diagnosed with HGSOC between 2010 and 2013 were obtained from Pathology Research Core in Mayo Clinic. A total of 60 FFPE blocks from 1 primary tumor and 2 metastatic sites of each patient were used to make the slides. Five MES-subtype biomarkers, including ACTA2, FAP, POSTN, COL5A1, and VCAN, were selected for immunohistochemistry (IHC) to determine the protein localization and intensities. Two researchers scored tumor sections for intensity as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), and for localization (stroma/tumor, cytoplasm/nuclear). Statistical analysis was performed using GraphPad Prism 7 software and statistical differences were evaluated using t-test. Results: All 5 proteins evaluated exclusively localized in stroma rather than cancer cells (p&lt;0.001 for all 5 proteins). Only FAP showed significantly different staining intensity between MES and ANM subtype of OC patient samples (p= 0.0114). When comparing primary vs. metastatic sites, only COL5A1 showed significant difference (p=0.019). Conclusion: We report that 5 biomarkers traditionally associated with the MES subtype HGSOC arise from cancer-associated stroma instead of cancer cells. Considering the potential roles of these proteins in mediating HGSOC behavior, it may suggest “stroma targeting” as an alternative therapy to “cancer targeting” to improve OC patient outcomes. Although all 5 genes were reported as MES biomarkers in mRNA-based analysis, we found that FAP was the only protein differentially expressed between MES and ANM subtype suggesting it is a better characteristic biomarker for MES subtype. COL5A1 showed significance in staining between primary and metastatic cancer which may indicates the potential role of COL5A1 in metastasis in OC. Citation Format: Qing Zhang, Kimberly R. Kalli, Chen Wang, William Cliby. The mesenchymal subtype biomarkers arise from stroma rather than cancer cells in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4491.

Abstract Background: In ovarian cancer (OC), the intraperitoneal fibrosis that accompanies metastasis is a common scenario and associated with significant morbidity and gut dysfunction. Accumulating evidences indicate that Transforming Growth Factor β (TGF-β) signaling pathway plays a key role in cancer associated fibrosis and metastasis. Several TGF-β inhibitors, including Galunisertib (LY2157299) which is a TGF-β receptor I inhibitor, are currently in clinical trials for fibrosis-related diseases and cancer. Our hypothesis is that targeted anti-cancer therapies can block the growth and spread of cancer or specifically address the fibrosis accompanying metastatic peritoneal cancer. In this study, we investigated the efficacy of Galunisertib in inhibiting OC fibrosis and metastasis in vitro. Methods: Five OC cell lines, including SKOV3ip, HeyA8, OVCAR8, OV2008, and PEO4, and one cancer-associated fibroblast (CAF) cell line, TRS3, were tested in this study. We utilized a co-culture system that has successfully been used to study the interactions between cancer cells and local environment, including CAFs. Twenty-three candidate TGF-β signaling- and fibrosis-related genes were selected based on previous studies. RNA and protein from both fibroblast cells and OC cells were collected for RT-qPCR and western blot analysis. MTT assay, Wound healing assay and Matrigel invasion assay were used to study OC cell proliferation, migration and invasion. Chou-Talalay assays were performed to study the interactions between Galunisertib and Carboplatin and Paclitaxel. Results: RT-qPCR results showed that Galunisertib treatment suppresses the expression of both TGF-β receptor I and TGF-β1 which is the most abundant isoform of TGF-β in OC. Western blot results showed that Galunisertib treatment reverses the TGF-β1-induced SMAD2/3 phosphorylation, confirming the efficacy of Galunisertib in TGF-β signaling pathway blockage. Intriguingly, Galunisertib treatment inhibits the expression of candidate fibrosis-related genes, like ACTA2, TIMP3, COL5A1, COL11A1, and VCAN in both OC and CAF cells. Would healing and Transwell invasion assay also demonstrated that Galunisertib treatment effectively reduces both basal and TGF-β1-induced cell migration and invasion (p&lt;0.05). MTT assay showed that Galunisertib is able to inhibit the proliferation of both OC and CAFs cells with IC50 values range from 100-400 μm. Additionally, Galunisertib shows strong synergistic effects with both Carboplatin and Paclitaxel in Chou-Talalay assays. Conclusion: Galunisertib treatment suppresses the expression of TGF-β-dependent fibrosis-related genes from OC cell lines and CAFs. Coincident with this inhibition of fibrosis related genes, galunisrtib reduces cell proliferation, migration, and invasion. These data suggest that targeting the TGF-β pathway is a viable therapeutic approach to reduce both symptoms related to OC-related fibrosis and cancer metastasis in OC. Citation Format: Qing Zhang, William A. Cliby. Galunisertib inhibits ovarian cancer cell fibrosis and metastasis in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3026.

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the US. Müllerian inhibiting substance (MIS) is a glycoprotein hormone that causes Müllerian duct regression through a complex of TGF- β family homologous receptors: a type II ligand-binding receptor (T2R) and a tissue-specific type I receptor (T1R) that initiates downstream signaling. MIS has been proposed as potential therapeutic agent in OC. We have previously shown that specific combination of T2R/T1R receptors are expressed on OC cells, however data is lacking on the MIS-dependent pathways activated in OC cells in a T1R-specific manner. These limitations hinder further development of MIS as a therapy. We sought to determine the downstream signaling pathways activated by MIS in a T1R specific manner. We designed a chimeric OC cell model system in which GM-CSF can function as the ligand to specifically activate candidate T2R/T1R combinations (MISRII with either ALK2, ALK3, or ALK6). RNA-Seq technology was used to identify differentially expressed early and late response genes. Over 15,032 genes were analyzed, restricted to genes in which there were &gt; 32 counts for duplicate samples. Pathway analysis was performed using Ingenuity's Integrative Pathway Analysis Tool (IPA) for genes which were expressed over 2 fold, with an False Discovery Rate (FDR)&lt;0.1 and log (Counts per Million, CPM)&gt;-0.001. We observed 56, 37, and 51 early response (1hr) genes which were up- or down-regulated after stimulation in OC cells engineered to express either ALK2, ALK3, and ALK6, respectively. We identified a subset of 18 of genes (e.g., ID1, SMAD7, FOSB) which were upregulated after treatment in all MISRII/T1R combinations, suggesting redundancy amongst the three T1R partner receptors. These observations will be confirmed by RT-qPCR and immunoblotting. A majority of these redundant genes are known to play roles in TGF-β signaling and cell proliferation pathways. Importantly, as we have previously shown that not all T1R candidates are expressed in OC, we report subsets of genes (e.g., RASSF6, C/EBPBβ, CSMD3) which were uniquely altered after stimulation only in the presence of a specific T1R. This indicates specific pathway activation is dependent upon T1R expression pattern in OC and likely determines response. In addition to previously known TGF- β signaling pathway, we found gene signatures (e.g. FOSB, RASSF6, LRP1B, ID1) suggesting pathways which affect cell proliferation, apoptosis, chemotherapy resistance, and DNA repair in response to MIS. This is the first study to begin to define the specific roles candidate T1R's play in signaling in response to MIS in an OC model. An understanding of these pathways is a necessary prerequisite to examining MIS as a therapy. These preliminary data suggest a role for MIS in apoptosis, cytotoxic drug resistance and DNA repair. This can be used to test combination therapy with currently available drugs and MIS in OC treatment. Citation Format: Qing Zhang, Eati Basal, Jaime I. Davila, Xueqian Yin, Edward B. Leof, William A. Cliby. Comparative RNA-Seq analysis of MIS signaling: Potential relevance as therapeutic strategy in ovarian cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2014-3462

Abstract Recent RNA-sequencing studies have identified variant transcripts, fusion transcripts and novel transcripts in cancer. However, little is known about the role of these variant transcripts in tumor development. To identify a specific role of these variant transcripts in tumor development toward chemotherapy resistance, we generated a custom retroviral cDNA expression library from 10 ovarian tumors with resistance to chemotherapy. Retrovirues were generated from the custom library, and they were used to transduced exogenous genes into ovarian cancer cell line A1847, a cell line chosen for its sensitivity to paclitaxel. A1847 cells transduced with retroviruses carrying alkaline phosphatase was used as a control. Transuced cells were selected with 25 nM paclitaxel. 87 clones survived the selection in library-tranduced cells whereas none survived the selection in alkaline phosphatase-tranduced control cells. Forty seven clones survive the secondary screens. Sequence analysis of transduced genes in these surviving clones indicate that 4 paclitaxel-resistant clones contain RABL3 splice variant. Although 195 accessions corresponding to wildtype transcripts are reported in AceView, there is a single accession corresponding to RABL splice variant that we identified in this screen. RNAi-mediated downregulation of RABL3 resulted in enhance sensitivity toward paclitaxel in these resistant clones. Ectopic expression of RABL3 splice variant resulted in increased resistant to paclitaxel. Collectively, these results demonstrate the feasibility of performing functional genetic screens to identify novel splice variants involved in tumor development. 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 5120. doi:1538-7445.AM2012-5120