Abstract
Background: Prospective clinical trials stratifying by PIK3CA genotype is necessary to determine if the mutation predicts for increased sensitivity to agents targeting the phosphoinositide ...3-kinase (PI3K) pathway. It is crucial to select appropriate assays to optimize operational, scientific and timeline requirements for patient enrollment in a real world setting. Here we report application of three different PIK3CA mutation test methods during the course of a PI3K TKI development.
Methods: We customized Sanger sequencing, cobas® assay and a proprietary investigational use only (IUO) assay to detect point mutations in PIK3CA in order to accommodate the development needs at the different stages of clinical development of PIK3CA TKIs. We performed method comparison studies to determine the most cost effective and “fit-for-purpose” assay for clinical trial testing. Specimen requirements, assay performance characteristics, turnaround-time (TAT) and valid result rates were assessed to select the best test method for patient stratification to meet the requirements of each stage of drug development.
Results: PIK3CA test results can be obtained from a single, 5μm FFPE section with ≥0.1% and ≥10% tumor in the IUO and cobas® tests respectively. Sanger Sequencing requires longer processing time and ≥30% tumor content. The cobas® assays were validated in FFPE samples and can reliably detect PIK3CA mutations in exons 9 and 20 with ≥5% mutation level. The method comparison study showed that the concordance rate between Sanger sequencing and cobas® PIK3CA assay is 86% among the 360 samples tested while it is 96% between the cobas® assay and the IUO assay among 395 samples tested. The IUO assay is slightly more sensitive, and calls out the exact mutation while the cobas® assay may not. The Sanger sequencing demonstrated the lowest sensitivity and required more DNA input, which resulted in more samples failing to yield a valid result. The cobas® assay is a more user-friendly assay allowing better laboratory efficiency and TAT. Patients selected using the IUO assay showed a median progression-free survival nearly twice as long among those receiving a PI3K TKI compared to placebo.
Conclusions: The most important factor in selecting a testing platform for clinical trial patient stratification is the assay performance characteristics. We employed the Sanger sequencing assay in the initial trial testing, then switched to the cobas® assay, and finally to the IUO assay to meet the requirements as the drug progressed through different stages of clinical development. The IUO assay has the performance characteristics that meet the regulatory requirements and a validated clinical utility in selecting patients for PI3K TKI treatment, thus on its way to a companion diagnostic submission.
Citation Format: Hua C. Gong, Sharmila Manjeshwar, Violet Abraham, Shawn Rivera. “Fit-for-purpose” PIK3CA assays for patient stratification in breast cancer clinical trials abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3976.
Background Q-F (NCT02668653) showed that the highly potent, selective, type 2 FLT3 inhibitor quizartinib (Q) + standard chemotherapy ± transplantation, followed by Q monotherapy for ≥36 cycles, ...reduced the relative risk of death by 22.4% vs placebo (P) in newly diagnosed (nd) FLT3-ITD+ AML, with HR of 0.776 and P value of 0.0324 (PMID: 37116523). In Q-F, FLT3-ITD mutation status was determined using a FLT3-ITD mutation detection clinical trial assay (CTA) validated under design control by Navigate BioPharma Services, Inc. We describe the results of the bridging study, aimed to show agreement between the CTA & the LeukoStrat CDx FLT3 Mutation Assay (CDx, by Invivoscribe) in FLT3-ITD+ pt selection and to determine if Q efficacy (overall survival OS) was maintained in nd FLT3-ITD+ AML pts from Q-F, if pts had been selected using the CDx. Methods In both CTA & CDx, DNA, extracted from bone marrow (n=884 each) or peripheral blood (n=139 each), was amplified via PCR and amplicons were detected via capillary electrophoresis. A sample was considered CTA+ if the variant allele frequency ( FLT3-ITD/total FLT3) was ≥3% and CDx+ if the signal ratio (SR; FLT3-ITD/ FLT3 WT) was ≥0.05. The agreement between CTA & CDx was based on evaluating CTA+ & CTA− samples with the CDx assay. A primary analysis included the CDx detected (CDx+ & CDx−) and the CDx invalid results. A secondary analysis used CDx+ and CDx− results only. To establish agreement between CDx & CTA, positive % agreement (PPA) and negative % agreement (NPA) were determined using CTA results as reference for the agreement analysis set (AAS). Concordance was established if the lower bounds of the 95% CIs for both PPA & NPA exceeded 90% for the analysis that included the invalid CDx results. Median OS in the subgroups was calculated based on Kaplan-Meier estimates. Stratified Cox proportional hazards regression model was used to estimate HRs, 95% CI, and P value. Results Full analysis set (N=3468) included all Q-F screened CTA+ pts (n=863), all screened CTA− pts (n=2556), and pts with unknown CTA status not eligible for randomization due to other criteria (n=49). Of these, 1032 pts formed the primary analysis set (PAS), including all pts randomized in Q-F with samples available for CDx testing (N=513: Q, n=254; P, n=259) and a randomly selected subset of CTA− pts (n=519). The ascertainment rate was 95.2% (513/539), as 26 of the 539 pts randomized in Q-F were excluded from the bridging study. Within the PAS, 3 samples were not tested by CDx due to insufficient volume/DNA amount. The AAS (N=1029: CTA+, n=513; CTA−, n=516) included pts in the PAS with valid CTA results and tested with CDx. In the AAS, 6 samples (3 CTA+, 3 CTA−) did not yield valid CDx results, resulting in 1023 CDx-evaluable total pts (CTA+, n=510; CTA−, n=513). Among 510 CTA+ samples, 483 were CDx+. Among 513 CTA− samples, 513 were CDx−. Therefore, 996 samples yielded concordant results, 27 samples yielded discordant results, and 6 samples did not yield a valid CDx result for comparison. Point estimates of PPA & NPA were 94.2% & 99.4%, respectively, with invalid CDx results included in the calculation, and 94.7% & 100%, respectively, without invalid CDx results. The lower bounds of the 95% CIs were all above the corresponding acceptance criterion of 90% for PPA & NPA (Table 1). In Q-F, the prevalence of CTA+ was 24.9% among screened pts (863/3468), whereas in the bridging study, 49.9% (510/1023) of pts were CTA+: this enrichment in CTA+ pts could lead to a biased estimate of the agreement between CDx & CTA when using CDx as reference. The positive predictive value (PPV) and negative predictive value (NPV) of the CDx adjusted for this enrichment ± invalid CDx results, showed that the lower bounds of the 95% CIs were all >95% (Table 1). The efficacy OS analysis in the intent-to-treat (ITT) CDx+ population (ITT CDx+=CTA+ & CDx+; N=483: Q, n=242; P, n=241) demonstrated a clinically relevant OS improvement with Q (median OS of 29.4 months) vs P (median OS of 14.8 months), resulting in 14.6 months prolongation of median OS, with an HR of 0.794 (95% CI 0.621-1.014), corresponding to a 20.6% reduction in relative risk of death (Figure 1). Conclusions This study showed 1) agreement between CDx & CTA in identifying nd FLT3-ITD+ AML pts and 2) that OS benefit provided by Q in the ITT CDx+ population is comparable with the OS benefit in the ITT population of Q-F. The LeukoStrat CDx FLT3 Mutation Assay aids in assessing AML pts for Q therapy.
Any complete account of moral action and choice requires an account of the character of moral agents. In particular, it requires an account of the role of virtue in moral agency. Virtue theory ...provides responses to four types of objections to deontological and consequentialist theories of right action. First, the model of deliberation available to these views does not explain how agents deliberate to a moral choice. Second, the justification that agents would offer under deontological and consequentialist accounts is one that alienates agents from connections and concerns for particular others. Third, complete satisfaction of deontological and consequentialist moral requirements may demand that agents relinquish what they most care about. Fourth, agents who attempt to help others by utilizing general moral principles alone may undermine or dishonor the autonomy of those they attempt to help. The objections illuminate central questions about agency but they do not show that the theories are inadequate as theories of right action. While virtue theory is sometimes presented by its defenders as a full-fledged rival to deontological and consequentialist theory, I argue that a plausible version of virtue theory must retain a key element typically taken to characterize deontological theory: a role for general justifying principles. General principles will not be enough to account for agency, however. The true moral theory must presuppose an account of the virtues in order to complete its account of moral deliberation and choice.
Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend ...on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to “induced” TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies.
Display omitted
Display omitted
•Distinct epigenetic state enables glioblastoma (GBM) cells to propagate tumors in vivo•Four TFs reprogram differentiated GBM cells into tumor-propagating stem-like cells•These four TFs are coordinately expressed in stem-like cells in primary human tumors•The TF target LSD1 may serve as a therapeutic target in tumor-propagating cells
A regulatory network requiring four transcription factors is sufficient to reprogram differentiated glioblastoma tumor cells to tumor-propagating stem-like cells. Characterization of this network reveals potential therapeutic targets for eradicating tumor-propagating cells, which are resistant to current therapies.
Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to ...addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.
Glioblastoma (GBM) is thought to be driven by a subpopulation of cancer stem cells (CSCs) that self-renew and recapitulate tumor heterogeneity yet remain poorly understood. Here, we present a ...comparative analysis of chromatin state in GBM CSCs that reveals widespread activation of genes normally held in check by Polycomb repressors. These activated targets include a large set of developmental transcription factors (TFs) whose coordinated activation is unique to the CSCs. We demonstrate that a critical factor in the set, ASCL1, activates Wnt signaling by repressing the negative regulator DKK1. We show that ASCL1 is essential for the maintenance and in vivo tumorigenicity of GBM CSCs. Genome-wide binding profiles for ASCL1 and the Wnt effector LEF-1 provide mechanistic insight and suggest widespread interactions between the TF module and the signaling pathway. Our findings demonstrate regulatory connections among ASCL1, Wnt signaling, and collaborating TFs that are essential for the maintenance and tumorigenicity of GBM CSCs.
Display omitted
•Epigenomic profiles of glioblastoma stem cells and comparators•An aberrant network of developmental transcription factors in cancer stem cells•ASCL1 is essential for glioblastoma stem cell maintenance and tumorigenicity•ASCL1 activates Wnt signaling by directly repressing the negative regulator DKK1
Glioblastoma brain tumors contain a highly tumorigenic subpopulation of cells with stem-like features. Using epigenomic profiling, Chi, Bernstein, and colleagues identify a set of developmental transcription factors normally repressed by Polycomb complexes that become activated in these stem-like cancer cells. One of these factors, ASCL1, is shown to be essential for glioblastoma stem cell maintenance and tumorigenicity and to function as an activator of Wnt signaling in this setting.
The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by ...directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.
Display omitted
•A single aberrant transcription factor directly activates or represses enhancers•Chromatin remodeling at enhancer elements is dictated by the underlying DNA sequence•EWS-FLI1 displays divergent patterns of chromatin remodeler recruitment•De novo enhancers mediate tumor dependencies
Using integrated analyses of chromatin states in human Ewing sarcoma, Riggi et al. find that the EWS-FLI1 fusion protein drives an oncogenic regulatory program governing both survival and differentiation by either creating enhancers de novo or competing with endogenous ETS to repress conserved enhancers.
Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer-associated ...fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.
Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME). We dissected these influences in isocitrate ...dehydrogenase (IDH)-mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses.
Environmental change is intensifying the biodiversity crisis and threatening species across the tree of life. Conservation genomics can help inform conservation actions and slow biodiversity loss. ...However, more training, appropriate use of novel genomic methods and communication with managers are needed. Here, we review practical guidance to improve applied conservation genomics. We share insights aimed at ensuring effectiveness of conservation actions around three themes: (1) improving pedagogy and training in conservation genomics including for online global audiences, (2) conducting rigorous population genomic analyses properly considering theory, marker types and data interpretation and (3) facilitating communication and collaboration between managers and researchers. We aim to update students and professionals and expand their conservation toolkit with genomic principles and recent approaches for conserving and managing biodiversity. The biodiversity crisis is a global problem and, as such, requires international involvement, training, collaboration and frequent reviews of the literature and workshops as we do here.
PDF
