Harnessing imaging tools to guide immunotherapy trials: summary from the National Cancer Institute Cancer Imaging Steering Committee workshop.

As the immuno-oncology field continues the rapid growth witnessed over the past decade, optimising patient outcomes requires an evolution in the current response-assessment guidelines for phase 2 and 3 immunotherapy clinical trials and clinical care. Additionally, investigational tools-including image analysis of standard-of-care scans (such as CT, magnetic resonance, and PET) with analytics, such as radiomics, functional magnetic resonance agents, and novel molecular-imaging PET agents-offer promising advancements for assessment of immunotherapy. To document current challenges and opportunities and identify next steps in immunotherapy diagnostic imaging, the National Cancer Institute Clinical Imaging Steering Committee convened a meeting with diverse representation among imaging experts and oncologists to generate a comprehensive review of the state of the field.

Considerations on Integrating Prostate-Specific Membrane Antigen Positron Emission Tomography Imaging Into Clinical Prostate Cancer Trials by National Clinical Trials Network Cooperative Groups.

PURPOSE: As prostate-specific membrane antigen (PSMA) positron emission tomography (PET) becomes increasingly available in the United States, the greater sensitivity of the technology in comparison to conventional imaging poses challenges for clinical trials. The NCI Clinical Imaging Steering Committee (CISC) PSMA PET Working Group was convened to coordinate the identification of these challenges in various clinical scenarios and to develop consensus recommendations on how best to integrate PSMA PET into ongoing and upcoming National Clinical Trials Network (NCTN) trials. METHODS: NCI CISC and NCI Genitourinary Steering Committee members and leadership nominated clinicians, biostatisticians, patient advocates, and other imaging experts for inclusion in the PSMA PET Working Group. From April to July 2021, the working group met independently and in conjunction with the CISC to frame challenges, including stage migration, response assessment, trial logistics, and statistical challenges, and to discuss proposed solutions. An anonymous, open-ended survey was distributed to members to collect feedback on challenges faced. Representatives from each NCTN group were invited to present an overview of affected trials. From these discussions, the consensus document was developed and circulated for the inclusion of multiple rounds of feedback from both the Working Group and CISC. RESULTS: The current consensus document outlines the key challenges for clinical prostate cancer trials resulting from the increasing availability of PSMA PET. We discuss implications for patient selection and definition of end points and provide guidance and potential solutions for different clinical scenarios, particularly with regard to best practices in defining eligibility criteria and outcome measures. RECOMMENDATIONS: This article provides guidance regarding clinical trial design and conduct, and the interpretation of trial results.

Factors Affecting Combination Trial Success (FACTS): Investigator Survey Results on Early-Phase Combination Trials.

Experimental therapeutic oncology agents are often combined to circumvent tumor resistance to individual agents. However, most combination trials fail to demonstrate sufficient safety and efficacy to advance to a later phase. This study collected survey data on phase 1 combination therapy trials identified from ClinicalTrials.gov between January 1, 2003 and November 30, 2017 to assess trial design and the progress of combinations toward regulatory approval. Online surveys (N = 289, 23 questions total) were emailed to Principal Investigators (PIs) of early-phase National Cancer Institute and/or industry trials; 263 emails (91%) were received and 113 surveys completed (43%). Among phase 1 combination trials, 24.9% (95%CI: 15.3%, 34.4%) progressed to phase 2 or further; 18.7% (95%CI: 5.90%, 31.4%) progressed to phase 3 or regulatory approval; and 12.4% (95%CI: 0.00%, 25.5%) achieved regulatory approval. Observations of "clinical promise" in phase 1 combination studies were associated with higher rates of advancement past each milestone toward regulatory approval (cumulative OR = 11.9; p = 0.0002). Phase 1 combination study designs were concordant with Clinical Trial Design Task Force (CTD-TF) Recommendations 79.6% of the time (95%CI: 72.2%, 87.1%). Most discordances occurred where no plausible pharmacokinetic or pharmacodynamic interactions were expected. Investigator-defined "clinical promise" of a combination is associated with progress toward regulatory approval. Although concordance between study designs of phase 1 combination trials and CTD-TF Recommendations was relatively high, it may be beneficial to raise awareness about the best study design to use when no plausible pharmacokinetic or pharmacodynamic interactions are expected.

Seamless Designs: Current Practice and Considerations for Early-Phase Drug Development in Oncology.

Traditionally, drug development has evaluated dose, safety, activity, and comparative benefit in a sequence of phases using trial designs and endpoints specifically devised for each phase. Innovations in drug development seek to consolidate the phases and rapidly expand accrual with "seamless" trial designs. Although consolidation and rapid accrual may yield efficiencies, widespread use of seamless first-in-human (FiH) trials without careful consideration of objectives, statistical analysis plans, or trial oversight raises concerns. A working group formed by the National Cancer Institute convened to consider and discuss opportunities and challenges for such trials as well as encourage responsible use of these designs. We reviewed all abstracts presented at American Society of Clinical Oncology annual meetings from 2010 to 2017 for FiH trials enrolling at least 100 patients. We identified 1786 early-phase trials enrolling 57 559 adult patients. Fifty-one of the trials (2.9%) investigated 50 investigational new drugs, were seamless, and accounted for 14.6% of the total patients. The seamless trials included a median of 3 (range = 1-13) expansion cohorts. The overall risk of clinically significant treatment-related adverse events (grade 3-4) was 49.1% (range = 0.0-100%), and seven studies reported at least one toxic death. Rapid expansion of FiH trials may lead to earlier drug approval and corresponding widespread patient access to active therapeutics. Nevertheless, seamless designs must adhere to established ethical, scientific, and statistical standards. Protocols should include prospectively planned analyses of efficacy in disease- or biomarker-defined cohorts of sufficient rigor to support accelerated approval.

Challenges and Opportunities in Adapting Clinical Trial Design for Immunotherapies.

Immunotherapy adds an exciting new dimension to the treatment of cancer, joining other approaches as a key pillar in the oncotherapeutics armamentarium. Immuno-oncology agents harbor unique mechanisms of antitumor activity by leveraging the host immune system, which may result in response patterns, resistance kinetics, and toxicity profiles that differ from other systemic therapies. These features have led to many discussions on ways to optimally integrate immunotherapy into cancer clinical trials. This overview provides an introduction to the four CCR Focus articles that ensue, with special thoughts paid to clinical trial endpoints, biomarker development and validation, combination strategies, and limitations that arise with increasing use of these agents. In addition, this overview examines design concepts that may be applied to invigorate clinical trials and to maximize their impact in the immuno-oncology era.

Peroxisome-proliferator-activated receptor gamma coactivator 1 α contributes to dysmyelination in experimental models of Huntington's disease.

The peroxisome-proliferator-activated receptor gamma coactivator 1 α (PGC1α) has been implicated in the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Recent data demonstrating white matter abnormalities in PGC1α knock-out (KO) mice prompted us to examine the role of PGC1α in CNS myelination and its relevance to HD pathogenesis. We found deficient postnatal myelination in the striatum of PGC1α KO mice, accompanied by a decrease in myelin basic protein (MBP). In addition, brain cholesterol, its precursors, and the rate-limiting enzymes for cholesterol synthesis, HMG CoA synthase (HMGCS1) and HMG CoA reductase (HMGCR), were also reduced in PGC1α KO mice. Moreover, knockdown of PGC1α in oligodendrocytes by lentiviral shRNA led to a decrease in MBP, HMGCS1, and Hmgcr mRNAs. Chromatin immunoprecipitations revealed the recruitment of PGC1α to MBP promoter in mouse brain, and PGC1α over-expression increased MBP and SREBP-2 promoter activity, suggesting that PGC1α regulates MBP and cholesterol synthesis at the transcriptional level. Importantly, expression of mutant huntingtin (Htt) in primary oligodendrocytes resulted in decreased expression of PGC1α and its targets HmgcS1, Hmgcr, and MBP. Decreased expression of MBP and deficient myelination were found postnatally and in adult R6/2 mouse model of HD. Diffusion tensor imaging detected white matter abnormalities in the corpus callosum of R6/2 mice, and electron microscopy revealed thinner myelin sheaths and increased myelin periodicity in BACHD [bacterial artificial chromosome (BAC)-mediated transgenic model for Huntington's disease] mice expressing full-length mutant Htt. Together, these data suggest that PGC1α plays a role in postnatal myelination and that deficient PGC1α activity in oligodendrocytes may contribute to abnormal myelination in HD.

SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis.

Huntington's disease (HD), an incurable neurodegenerative disorder, has a complex pathogenesis including protein aggregation and the dysregulation of neuronal transcription and metabolism. Here, we demonstrate that inhibition of sirtuin 2 (SIRT2) achieves neuroprotection in cellular and invertebrate models of HD. Genetic or pharmacologic inhibition of SIRT2 in a striatal neuron model of HD resulted in gene expression changes including significant down-regulation of RNAs responsible for sterol biosynthesis. Whereas mutant huntingtin fragments increased sterols in neuronal cells, SIRT2 inhibition reduced sterol levels via decreased nuclear trafficking of SREBP-2. Importantly, manipulation of sterol biosynthesis at the transcriptional level mimicked SIRT2 inhibition, demonstrating that the metabolic effects of SIRT2 inhibition are sufficient to diminish mutant huntingtin toxicity. These data identify SIRT2 inhibition as a promising avenue for HD therapy and elucidate a unique mechanism of SIRT2-inhibitor-mediated neuroprotection. Furthermore, the ascertainment of SIRT2's role in regulating cellular metabolism demonstrates a central function shared with other sirtuin proteins.

Simvastatin induces cell death in a mouse cerebellar slice culture (CSC) model of developmental myelination.

Statins (inhibitors of HMG-CoA reductase) have shown promise in treating multiple sclerosis (MS). However, their effect on oligodendrocyte remyelination of demyelinated axons has not been clarified. Since developmental myelination shares many features with the remyelination process, we investigated the effect of lipophilic simvastatin on developmental myelination in organotypic cerebellar slice cultures (CSC). In this study, we first characterized developmental myelination in CSC from postnatal day (P)5 and P10 mice that express enhanced green fluorescence protein (eGFP) in oligodendrocyte-lineage cells. We then examined the effect of simvastatin on three developmental myelination stages: early myelination (P5 CSC, 2DIV), late myelination (P10 CSC, 2DIV) and full myelination (P10 CSC, 10DIV). We found that treatment with simvastatin (0.1 microM) for 6 days decreased the survival of Purkinje cells and oligodendrocytes drastically during the early myelination stage, while moderately during the late and full myelination stages. Oligodendrocytes are more resistant than Purkinje cells. The toxic effect of simvastatin could be rescued by the product of HMG-CoA reductase mevalonate but not low-density lipoprotein (LDL). Additionally, this toxic effect is independent of isoprenylation since farnesyl pyrophosphate (Fpp) but not geranylgeranyl pyrophosphate (GGpp) provided partial rescue. Our findings therefore suggest that inhibition of cholesterol synthesis is detrimental to neuronal tissue.

15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells.

BACKGROUND: Prostaglandin (PG) production is associated with inflammation, a major feature in multiple sclerosis (MS) that is characterized by the loss of myelinating oligodendrocytes in the CNS. While PGs have been shown to have relevance in MS, it has not been determined whether PGs have a direct effect on cells within the oligodendrocyte lineage. METHODS: Undifferentiated or differentiated mouse oligodendrocyte precursor (mOP) cells were treated with PGE2, PGF2alpha, PGD2 or 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). Cell growth and survival following treatment were examined using cytotoxicity assays and apoptosis criteria. The membrane receptors for PGD2 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma, as well as reactive oxygen species (ROS) in the death mechanism were examined. RESULTS: PGE2 and PGF2alpha had minimal effects on the growth and survival of mOP cells. In contrast, PGD2 and 15d-PGJ2 induced apoptosis of undifferentiated mOP cells at relatively low micromolar concentrations. 15d-PGJ2 was less toxic to differentiated mOP cells. Apoptosis was independent of membrane receptors for PGD2 and the nuclear receptor PPARgamma. The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels. However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or alpha-tocopherol. CONCLUSION: Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.

New mouse oligodendrocyte precursor (mOP) cells for studies on oligodendrocyte maturation and function.

Oligodendrocyte precursor (OP) cells give rise to mature oligodendrocytes (OL), which are necessary for myelination of axons during CNS development and following damage to the myelin sheath that occurs in demyelinating diseases. To facilitate studies designed to understand OP maturation and OL function, we have developed OP cells that can be grown continuously, expanded, and differentiated into mature OLs. Cultures of late passage mOP cells grown in proliferation medium are highly pure early stage oligodendrocyte precursors where > 90% assume a characteristic bipolar morphology. Immunocytochemical analysis using antibodies that recognize progressive stages of OP maturation (A2B5, NG2, GD3 and O4) confirmed that mOP cells have a stable early stage OP cell phenotype. In addition, mOP cells can be induced to differentiate into mature forms of oligodendrocytes in vitro and in vivo, as characterized morphologically by the presence of multiple processes with secondary and tertiary branches, and by immunostaining and quantitative real-time PCR for the mature oligodendrocyte markers MBP, MAG, PLP, and MOBP. Finally, differentiation of mOP cells was accompanied by up-regulation of mRNA encoding Olig2 but not Olig1, which is consistent with previous findings showing that Olig2 is necessary for specification of oligodendrocytes. These new mOP cells should significantly benefit in vitro and in vivo studies on OP maturation and function.

Detection of myelination using a novel histological probe.

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease.

Dual control of neurogenesis by PC3 through cell cycle inhibition and induction of Math1.

Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.

Initiation and maintenance of CNTF-Jak/STAT signaling in neurons is blocked by protein tyrosine phosphatase inhibitors.

Cytokines, including interferon-gamma and ciliary neurotrophic factor (CNTF), act in common through tyrosine kinase-based Jak/STAT signaling pathways. We found that activation of the Jak/STAT pathway by both interferon-gamma and CNTF in nerve cells was rapidly terminated by tyrosine phosphatase inhibitors. Exposure of human neuroblastoma cells, BE(2)-C, first to tyrosine phosphatase inhibitors (either phenylarsine oxide or PTP inhibitor-2) prevented Jak1, STAT1 and STAT3 activation elicited subsequently by either CNTF or interferon-gamma. In contrast, exposure of these cells to phosphatase inhibitors after initial stimulation by CNTF or interferon-gamma prevented the normal time-dependent decrease of total cellular phosphotyrosine-STAT levels as expected, while excluding already formed phosphotyrosine-STAT from the nucleus. Thus, treatment of nerve cells with a tyrosine phosphatase inhibitor blocked nuclear signal transduction. A similar inhibition of CNTF-Jak/STAT signaling was observed following tyrosine phosphatase inhibition in SH-SY5Y human neuroblastoma cells, HMN-1 mouse motor neuron-neuroblastoma hybrid cells, HepG2 human hepatoma cells and embryonic chick ciliary ganglion and retinal neurons. Expression of dominant-negative forms of the tyrosine phosphatases, SHP-1 and/or SHP-2, in BE(2)-C cells had no effect on CNTF activation of STAT or on the ability of phosphatase inhibitors to block signaling. Further, results from H-35 cells expressing gp130 receptor subunits lacking functional SHP-2 binding sites revealed normal cytokine activation of Jak and STAT that was inhibited by phosphatase inhibitors. These findings suggest a critical control for regulating the initiation of Jak/STAT signaling requiring tyrosine phosphatase activity.