Acute Myeloid Leukemia Case after Gene Therapy for Sickle Cell Disease.

Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the ßA-T87Q-globin gene. Acute myeloid leukemia developed in a woman approximately 5.5 years after she had received LentiGlobin for sickle cell disease as part of the initial cohort (Group A) of the HGB-206 study. An analysis of peripheral-blood samples revealed that blast cells contained a BB305 lentiviral vector insertion site. The results of an investigation of causality indicated that the leukemia was unlikely to be related to vector insertion, given the location of the insertion site, the very low transgene expression in blast cells, and the lack of an effect on expression of surrounding genes. Several somatic mutations predisposing to acute myeloid leukemia were present after diagnosis, which suggests that patients with sickle cell disease are at increased risk for hematologic malignant conditions after transplantation, most likely because of a combination of risks associated with underlying sickle cell disease, transplantation procedure, and inadequate disease control after treatment. (Funded by Bluebird Bio.).

OncoThreads: visualization of large-scale longitudinal cancer molecular data.

MOTIVATION: Molecular profiling of patient tumors and liquid biopsies over time with next-generation sequencing technologies and new immuno-profile assays are becoming part of standard research and clinical practice. With the wealth of new longitudinal data, there is a critical need for visualizations for cancer researchers to explore and interpret temporal patterns not just in a single patient but across cohorts. RESULTS: To address this need we developed OncoThreads, a tool for the visualization of longitudinal clinical and cancer genomics and other molecular data in patient cohorts. The tool visualizes patient cohorts as temporal heatmaps and Sankey diagrams that support the interactive exploration and ranking of a wide range of clinical and molecular features. This allows analysts to discover temporal patterns in longitudinal data, such as the impact of mutations on response to a treatment, for example, emergence of resistant clones. We demonstrate the functionality of OncoThreads using a cohort of 23 glioma patients sampled at 2-4 timepoints. AVAILABILITY AND IMPLEMENTATION: Freely available at http://oncothreads.gehlenborglab.org. Implemented in Java Script using the cBioPortal web API as a backend. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Proof of Concept for Biomarker-Guided Targeted Therapy against Ovarian Cancer Based on Patient-Derived Tumor Xenografts.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Genetic heterogeneity and clonal evolution during metastasis in breast cancer patient-derived tumor xenograft models.

Genetic heterogeneity within a tumor arises by clonal evolution, and patients with highly heterogeneous tumors are more likely to be resistant to therapy and have reduced survival. Clonal evolution also occurs when a subset of cells leave the primary tumor to form metastases, which leads to reduced genetic heterogeneity at the metastatic site. Although this process has been observed in human cancer, experimental models which recapitulate this process are lacking. Patient-derived tumor xenografts (PDX) have been shown to recapitulate the patient's original tumor's intra-tumor genetic heterogeneity, as well as its genomics and response to treatment, but whether they can be used to model clonal evolution in the metastatic process is currently unknown. Here, we address this question by following genetic changes in two breast cancer PDX models during metastasis. First, we discovered that mouse stroma can be a confounding factor in assessing intra-tumor heterogeneity by whole exome sequencing, thus we developed a new bioinformatic approach to correct for this. Finally, in a spontaneous, but not experimental (tail-vein) metastasis model we observed a loss of heterogeneity in PDX metastases compared to their orthotopic "primary" tumors, confirming that PDX models can faithfully mimic the clonal evolution process undergone in human patients during metastatic spreading.

Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia.

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19- relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Correction of copy number induced false positives in CRISPR screens.

Cell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function viability screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70-80% decrease of false positive hits with either method in regions of high copy number compared to no correction.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Genomic Analysis of Nasopharyngeal Carcinoma Reveals TME-Based Subtypes.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 (CDKN2A) locus and mutations in NF-κB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-naïve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFß pathway activation signature.Implications: This study represents the first integrated analysis of mutation, copy number, and gene expression data in NPC and suggests how tumor genetics and EBV infection influence the tumor microenvironment in this disease. These insights should be considered for guiding immunotherapy treatment strategies in this disease. Mol Cancer Res; 15(12); 1722-32. ©2017 AACR.

The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment.

Persistence of circulating endothelial microparticles in COPD despite smoking cessation.

INTRODUCTION: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. METHODS: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry. RESULTS: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. CONCLUSIONS: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. TRIAL REGISTRATION NUMBER: NCT00974064; NCT01776398.

Correlative Analysis of Genetic Alterations and Everolimus Benefit in Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results From BOLERO-2.

PURPOSE: To explore the genetic landscape of tumors from patients enrolled on the BOLERO-2 trial to identify potential correlations between genetic alterations and efficacy of everolimus treatment. The BOLERO-2 trial has previously demonstrated that the addition of everolimus to exemestane prolonged progression-free survival by more than twofold in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. PATIENTS AND METHODS: Next-generation sequencing was used to analyze genetic status of cancer-related genes in 302 archival tumor specimens from patients representative of the BOLERO-2 study population. Correlations between the most common somatic alterations and degree of chromosomal instability, and treatment effect of everolimus were investigated. RESULTS: Progression-free survival benefit with everolimus was maintained regardless of alteration status of PIK3CA, FGFR1, and CCND1 or the pathways of which they are components. However, quantitative differences in everolimus benefit were observed between patient subgroups defined by the exon-specific mutations in PIK3CA (exon 20 v 9) or by different degrees of chromosomal instability in the tumor tissues. CONCLUSION: The data from this exploratory analysis suggest that the efficacy of everolimus was largely independent of the most commonly altered genes or pathways in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. The potential impact of chromosomal instabilities and low-frequency genetic alterations on everolimus efficacy warrants further investigation.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

A distinct p53 target gene set predicts for response to the selective p53-HDM2 inhibitor NVP-CGM097.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.

Persistence of smoking-induced dysregulation of miRNA expression in the small airway epithelium despite smoking cessation.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/ß-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

Intraflagellar transport gene expression associated with short cilia in smoking and COPD.

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

Reduction of HBV replication prolongs the early immunological response to IFNα therapy.

BACKGROUND & AIMS: The interaction between HBV replication and immune modulatory effects mediated by IFNα therapy is not well understood. We characterized the impact of HBV DNA replication on the early IFNα-induced immunomodulatory mechanisms. METHODS: We interrogated the transcriptional, serum cytokine/chemokine and cellular immune profiles of 28 patients with HBeAg+ chronic HBV infection (CHB) randomly assigned to one of 4 treatment cohorts (untreated n=5, weekly dosing of 360 µg Pegasys [PegIFNα] n=11, daily dose of 300 mg Viread [tenofovir disoproxil fumarate, TDF] n=6, or a combination of both n=6). Samples were characterized at multiple early time points through day 14 of therapy, after which all patients were given standard of care (180 µg Pegasys injected subcutaneously, weekly). RESULTS: PegIFNα induced a distinct and rapid up-regulation of IFN signaling pathway that coincided with increase detection of distinct serum cytokines/chemokines (IL-15, IL-6, and CXCL-10) and the up-regulation of the frequency of proliferating NK and activated total CD8+ T cells. IFNα treatment alone did not result in rapid decay of HBV replication and was not able to restore the defective HBV-specific T cell response present in CHB patients. In addition, the IFNα immune-stimulatory effects diminished after the first dose, but this refractory effect was reduced in patients where HBV replication was simultaneously inhibited with TDF. CONCLUSIONS: We present here the first comprehensive description of the early effects of IFNα treatment on immune and viral biomarkers in HBeAg+ CHB patients. Our results show that PegIFNα-induced innate immune activation directly benefits from the suppression of HBV replication.