Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-cancer drugs enhances efficacy compared to the mono-therapy approach because it targets key pathways in a characteristically synergistic or an additive manner. This approach potentially reduces drug resistance, while simultaneously providing therapeutic anti-cancer benefits, such as reducing tumour growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. The 5-year survival rates for most metastatic cancers are still quite low, and the process of developing a new anti-cancer drug is costly and extremely time-consuming. Therefore, new strategies that target the survival pathways that provide efficient and effective results at an affordable cost are being considered. One such approach incorporates repurposing therapeutic agents initially used for the treatment of different diseases other than cancer. This approach is effective primarily when the FDA-approved agent targets similar pathways found in cancer. Because one of the drugs used in combination therapy is already FDA-approved, overall costs of combination therapy research are reduced. This increases cost efficiency of therapy, thereby benefiting the “medically underserved”. In addition, an approach that combines repurposed pharmaceutical agents with other therapeutics has shown promising results in mitigating tumour burden. In this systematic review, we discuss important pathways commonly targeted in cancer therapy. Furthermore, we also review important repurposed or primary anti-cancer agents that have gained popularity in clinical trials and research since 2012.

To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. .

Running title: Somatic genomic landscape of circulating tumor DNA Keywords: circulating tumor DNA, tumor heterogeneity, resistance, genomic landscape, cfDNA clonality Financial support: The study was funded by and conducted at Guardant Health, Inc. No additional grant support or administrative support was provided for the study. *Corresponding author:Stephen R. Fairclough Translational relevanceThis study describes genomic alterations from the largest cell-free circulating tumor DNA cohort to date, as derived from regular clinical practice. The high prevalence of resistance alterations found in advanced, treated cancer patients necessitated accurate methods for determining mutation clonality and driver/resistance status from plasma. We provide such methods, thereby extending the utility of cell-free DNA sequencing analysis. Our finding of an association between estimated circulating tumor DNA (ctDNA) levels and tumor mutational burden ascertained from plasma suggests that ctDNA level is likely an important variable to consider for immunotherapy applications of ctDNA analysis. Although cell-free DNA can provide a summary of tumor heterogeneity across multiple metastatic sites in a patient, our findings of high variability in ctDNA levels across patients, and its impact on variant detection, highlight the need for an improved understanding of factors influencing ctDNA levels and safe methods for maximizing them at the time of ctDNA testing. AbstractPurpose: Cell-free DNA (cfDNA) sequencing provides a non-invasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.Results: Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (TCGA and COSMIC;, with the principle differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR=5x10 -7 for EGFR and ERBB2), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel ...

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.

Cancer is a multi-stage process resulting from aberrant signaling pathways driving uncontrolled proliferation of transformed cells. The development and progression of cancer from a premalignant lesion towards a metastatic tumor requires accumulation of mutations in many regulatory genes of the cell. Different chemopreventative approaches have been sought to interfere with initiation and control malignant progression. Here we present research on dietary compounds with evidence of cancer prevention activity that highlights the potential beneficial effect of a diet rich in cruciferous vegetables. The Brassica family of cruciferous vegetables such as broccoli is a rich source of glucosinolates, which are metabolized to isothiocyanate compounds. Amongst a number of related variants of isothiocyanates, sulforaphane (SFN) has surfaced as a particularly potent chemopreventive agent based on its ability to target multiple mechanisms within the cell to control carcinogenesis. Anti-inflammatory, pro-apoptotic and modulation of histones are some of the more important and known mechanisms by which SFN exerts chemoprevention. The effect of SFN on cancer stem cells is another area of interest that has been explored in recent years and may contribute to its chemopreventive properties. In this paper, we briefly review structure, pharmacology and preclinical studies highlighting chemopreventive effects of SFN.

BackgroundBronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids.MethodsIn vitro studies were used for the dose–response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase).ResultsBoth compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0–80 μM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content.ConclusionsThe combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

Purpose Low dose metronomic (LDM) chemotherapy, combined with VEGF signaling pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth in many adult preclinical cancer models. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, a VEGF receptor inhibitor, in three pediatric extracranial solid tumor mouse models. Experimental Design In vitro dose–response study of topotecan and pazopanib was conducted on several neuroblastoma, osteosarcoma, and rhabdomyosarcoma cell lines. In vivo antitumor efficacies of the LDM topotecan and pazopanib as single agents and in combination were tested on 4 subcutaneous xenograft models and on 2 neuroblastoma metastatic models. Circulating angiogenic factors such as circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and microvessel densities were used as surrogate biomarker markers of antiangiogenic activity. Results In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, combination of topotecan + pazopanib (TP + PZ) showed significant antitumor activity and significant enhancement in survival compared with the respective single agents in all models. Reductions in viable CEP and/or CEC levels and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. Pharmacokinetic studies of both drugs did not reveal any drug–drug interaction. Conclusion Metronomic administration of TP + PZ showed a statistically significant antitumor activity compared with respective single agents in pediatric tumor mouse models and represent a valid option as a maintenance therapy in aggressive pediatric solid tumors.

We suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.