Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation.

Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation.

dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in ß-YAC mice.

Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over ß-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the -113A>G HPFH mutation. In a ß-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human ß-globin. The average -113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage-negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of ß-hemoglobinopathies.

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.

Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.

Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.

Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency.

To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.

Review: The Perioperative Use of Thromboelastography for Liver Transplant Patients.

Thromboelastography (TEG) is a viscoelastic test that allows rapid evaluation of clot formation and fibrinolysis from a sample of whole blood. TEG is increasingly utilized to guide blood product resuscitation in surgical patients and transfusions for liver transplant patients. Patients with severe liver failure have significant derangement of their clotting function due to impaired production of procoagulant and anticoagulant factors. Traditional coagulation studies are limited by the short time needed for the result and provide little information about the dynamics and strength of clot formation. In addition, traditional coagulation studies do not correlate well with bleeding episodes and may lead to over-transfusion of various blood products. Evidence is less robust regarding the use of TEG for transfusion management decisions in severe liver failure patients awaiting, undergoing, or immediately after liver transplant surgery. However, the available evidence suggests that systematic implementation of TEG rather than traditional coagulation studies results in the administration of fewer blood products without increased mortality or complications. The purpose of this study is to review the literature regarding the use of TEG in liver failure patients prior to liver transplant, intraoperatively, and postoperatively. Additional high-quality randomized controlled studies should be performed to evaluate the use of TEG to guide transfusion decisions, particularly in the postoperative period following liver transplantation.

Reactivation of γ-globin in adult ß-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Disorders involving ß-globin gene mutations, primarily ß-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human ß-globin locus-transgenic (ß-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from ß-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into ß-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ß- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Epigenomic Landscapes of hESC-Derived Neural Rosettes: Modeling Neural Tube Formation and Diseases.

We currently lack a comprehensive understanding of the mechanisms underlying neural tube formation and their contributions to neural tube defects (NTDs). Developing a model to study such a complex morphogenetic process, especially one that models human-specific aspects, is critical. Three-dimensional, human embryonic stem cell (hESC)-derived neural rosettes (NRs) provide a powerful resource for in vitro modeling of human neural tube formation. Epigenomic maps reveal enhancer elements unique to NRs relative to 2D systems. A master regulatory network illustrates that key NR properties are related to their epigenomic landscapes. We found that folate-associated DNA methylation changes were enriched within NR regulatory elements near genes involved in neural tube formation and metabolism. Our comprehensive regulatory maps offer insights into the mechanisms by which folate may prevent NTDs. Lastly, our distal regulatory maps provide a better understanding of the potential role of neurological-disorder-associated SNPs.

Methylome Analysis of Human Bone Marrow MSCs Reveals Extensive Age- and Culture-Induced Changes at Distal Regulatory Elements.

Human bone marrow stromal cells, or mesenchymal stem cells (BM-MSCs), need expansion prior to use as cell-based therapies in immunological and tissue repair applications. Aging and expansion of BM-MSCs induce epigenetic changes that can impact therapeutic outcomes. By applying sequencing-based methods, we reveal that the breadth of DNA methylation dynamics associated with aging and expansion is greater than previously reported. Methylation changes are enriched at known distal transcription factor binding sites such as enhancer elements, instead of CpG-rich regions, and are associated with changes in gene expression. From this, we constructed hypo- and hypermethylation-specific regulatory networks, including a sub-network of BM-MSC master regulators and their predicted target genes, and identified putatively disrupted signaling pathways. Our genome-wide analyses provide a broader overview of age- and expansion-induced DNA methylation changes and a better understanding of the extent to which these changes alter gene expression and functionality of human BM-MSCs.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies.

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices.

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

Effect of heterogeneous radio sensitivity on the survival, alpha beta ratio and biologic effective dose calculation of irradiated mammalian cell populations.

It is demonstrated that the surviving fraction of a population of cells with heterogeneous radio sensitivity, like that composing most malignant tumors, conforms to a different linear quadratic survival relation for dose less than about 3-5 Gy and dose greater than about 7-9 Gy. In the intermediate range of dose the survival relation for the population, as a whole, is not linear quadratic. Consequently, the value of the alpha beta ratio and the associated biologically effective dose calculation are different for the low and high dose range for most malignant tumors. Normal tissue cell populations responsible for organ function also have heterogeneous radio sensitivity, though to less degree than most malignant tumors. Consequently, the alpha beta ratio and associated biologically effective dose calculation related to the development of some acute early and chronic late developing radiation injuries are not the same in the low and high dose range. Variance of the distribution of α of a heterogeneous cell population lowers the effective value of the quadratic survival constant ß of the population, as a whole, and increases the α/ß ratio in the low dose range. Heterogeneous appearance of tumor cells (pleomorphism) and necrosis on biopsy or imaging studies reflect heterogeneity of the radio sensitivity of the cells. Greater heterogeneity implies a tendency to higher α/ß ratio. This may furnish a clinically accessible way to estimate a value of the α/ß ratio specific to an individual patient and tumor.

High-dose interleukin2 - a 10-year single-site experience in the treatment of metastatic renal cell carcinoma: careful selection of patients gives an excellent outcome.

BACKGROUND: VEGF-targeted therapy has become the mainstay of treatment for majority of mRCC patients. For most patients, benefit is short-lived and therefore treatment remains palliative in intent. HD IL2 is an effective immunotherapy treatment capable of durable remission in some patients but its unselected use has been difficult due to its modest response rate and considerable adverse effects. Using set pathology criteria as a selection tool in clinical practice, we have been able to show improved outcomes in our previous report. Here, we present an updated and extended report of this treatment and seek to explore any pathological, clinical and treatment variables likely to predict better outcomes. METHODS: This is an extension of a previously reported clinical audit, which includes mRCC cases treated with HD IL2 between 2003 and 2013. Since 2006, tumour specimens of potential candidates were routinely reviewed prospectively and stratified into Favourable or Other categories based on constitution of histological growth pattern, namely alveolar or solid versus papillary and/or sarcomatoid architecture; clear cell versus granular cell cytoplasmic morphology. HD IL2 was preferentially offered to patients with Favourable pathology. Outcome evaluation includes response rates, survival, and treatment tolerance. Multivariate analysis was performed to explore potential prognostic and predictive factors. RESULTS: Among prospectively selected patients with Favourable pathology (n = 106), overall response rate was 48.1 % (51/106) with CR rate of 21.6 % (23/106). Median OS was 58.1 months. Factors associated with significantly better response and/or survival includes favourable pathology pattern, higher cycle 1 tolerance and lower number of metastatic organ sites (<3). CAIX (Carbonic anhydrase 9) has prognostic value but is not predictive of response. Toxicities were those expected of IL2 but were manageable on general medical wards, with no treatment-related death. Importantly most complete responses were durable with 76 % (23/30) cases remained relapse-free (median 39 months follow up) and 2 of the seven who relapsed had had long-term disease free survival after resection of oligometastatic relapse. CONCLUSIONS: Our experience shows that HD IL2 remains an effective and safe treatment in well-selected cases of mRCC. The result in this single-institution patient series confirms similar outcomes to our previously reported retrospective series. Given the prospect of long-term remission, fit patients with Favourable histology and low disease burden should be considered for HD IL2 in an experienced centre. Better understanding has been gained from this in-depth analysis especially the examination of possible response predictors and strategies that can improve treatment outcome.

Extracting a low-dimensional description of multiple gene expression datasets reveals a potential driver for tumor-associated stroma in ovarian cancer.

Patterns in expression data conserved across multiple independent disease studies are likely to represent important molecular events underlying the disease. We present the INSPIRE method to infer modules of co-expressed genes and the dependencies among the modules from multiple expression datasets that may contain different sets of genes. We show that INSPIRE infers more accurate models than existing methods to extract low-dimensional representation of expression data. We demonstrate that applying INSPIRE to nine ovarian cancer datasets leads to a new marker and potential driver of tumor-associated stroma, HOPX, followed by experimental validation. The implementation of INSPIRE is available at http://inspire.cs.washington.edu .

Epigenetic Silencing of the Key Antioxidant Enzyme Catalase in Karyotypically Abnormal Human Pluripotent Stem Cells.

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells, however, the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase, a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues.

A randomized, double-blind phase II study evaluating cediranib versus cediranib and saracatinib in patients with relapsed metastatic clear-cell renal cancer (COSAK).

BACKGROUND: Preclinical work suggests SRC proteins have a role in the development of resistance to vascular endothelial growth factor (VEGF) targeted therapy in metastatic clear-cell renal cancer (mRCC). This hypothesis was tested in this trial using the SRC inhibitor saracatinib and the VEGF inhibitor cediranib. PATIENTS AND METHODS: Patients with disease progression after ≥1 VEGF-targeted therapy were eligible to participate in this double-blind, randomized (1:1) phase II study. The study compared the combination cediranib 30 mg once daily (o.d.) and saracatinib 175 mg o.d. (CS) (n = 69) or cediranib 45 mg o.d. and placebo o.d. (C) (n = 69). Archived tissue was used for biomarker analysis [SRC, focal adhesion kinase (FAK), von Hippel-Lindau, protein tyrosine phosphatase 1b and hypoxia-inducible factor 2α : n = 86]. The primary end point was progression-free survival (PFS) by RECIST v1.1. RESULTS: Between 2010 and 2012, 138 patients were randomized across 16 UK sites. The characteristics of the two groups were well balanced. Partial responses were seen in 13.0% for C and 14.5% for CS (P > 0.05). There was no significant difference in PFS [5.4 months (3.6-7.3 months) for C and 3.9 (2.4-5.3 months) for CS; hazard ratio (HR) 1.18 (0.94-1.48)] or overall survival (OS) [14.2 months (11.2-16.8 months) for C and 10.0 (6.7-13.2 months) for CS; HR 1.28 (1.00-1.63)]. There was no significant difference in the frequency of key adverse events, dose reductions or drug discontinuations. None of the biomarkers were prognostic for PFS or OS. FAK overexpression correlated with an OS benefit [HR 2.29 (1.09-4.82), P > 0.05], but not PFS, for CS. CONCLUSIONS: Saracatinib did not increase the efficacy of a VEGF-targeted therapy (cediranib) in this setting. Biomarker analysis did not identify consistent predictive biomarkers. CLINICALTRIALSGOV: NCT00942877.

Treatment patterns and clinical outcomes in patients with renal cell carcinoma in the UK: insights from the RECCORD registry.

BACKGROUND: The aim of the RECCORD registry was to gather real-world UK data on the use of targeted therapies in renal cell carcinoma (RCC) and assess clinical outcomes. Here, demographic and outcome data are presented with the treatment patterns and demographic profile of patients on the registry. PATIENTS AND METHODS: Patients were retrospectively identified at seven UK hospitals with large cancer centres in England (5), Scotland (1) and Wales (1). Anonymised data were collected through an online registry covering demographics, treatments and outcomes. Five hundred and fourteen UK adult patients with metastatic RCC were included in the study for analysis. Patients were included if they were treated for metastatic RCC at one of the seven centres, and started systemic anti-cancer treatment from March 2009 to November 2012 inclusive. In addition to demographic factors, the principal outcome measures were overall survival (OS), time to disease progression and toxicity. RESULTS: The majority of first-line treatment was with sunitinib; first-line use of pazopanib increased as the study progressed. 15.8% of patients received second-line treatment, half of whom were prescribed everolimus. Median OS (from initiation of first-line treatment) was 23.9 months (95% confidence interval [CI] 18.6-29.1 months), similar to that reported for clinical trials of targeted RCC therapies [Ljungberg B, Campbell SC, Choi HY et al. The epidemiology of renal cell carcinoma. Eur Urol 2011; 60: 615-621; Abe H, Kamai T. Recent advances in the treatment of metastatic renal cell carcinoma. Int J Urol 2013; 20: 944-955; Motzer RJ, Hutson TE, Tomczak P et al. Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 2009; 27: 3584-3590]. OS was significantly longer for those who received second-line treatment after disease progression (33.0 months; 95% CI 30.8-35.2 months) than those who did not (20.9 months; 95% CI 16.4-25.3 months; P = 0.008). CONCLUSIONS: RECCORD is a large 'real-world' database assessing metastatic RCC treatment patterns and outcomes. Treatment patterns changed over time as targeted therapies were approved and became widely available; survival data in RECCORD are consistent with those reported for systemic treatments in clinical trials. Kaplan-Meier analysis of results demonstrated that receiving second-line therapy was a major prognostic factor for longer OS.