Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed ...a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA).
An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping.
NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification.
Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.
Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST ...patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.
To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to ...monotherapies.
BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt
mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen-reactive IFNγ-producing CD8
T cells was evaluated by ELISpot assay.
NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor-bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8
T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8
T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy.
These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors.
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Pharmacological inhibitors of MAPK pathways English, Jessie M.; Cobb, Melanie H.
Trends in Pharmacological Sciences,
2002, 2002-Jan, 2002-1-00, 20020101, Letnik:
23, Številka:
1
Book Review, Journal Article
Recenzirano
Mitogen-activated protein kinases MAPKs, also called extracellular signal-regulated kinases (ERKs) are constituents of numerous signal transduction pathways, and are activated by protein kinase ...cascades. Intense efforts are under way to develop and evaluate compounds that target components of MAPK pathways. In this article, the current status of inhibitors of MAPK pathways will be presented with a focus on the properties of small-molecule inhibitors of p38, MEK1 and MEK2 protein kinases. Several of these inhibitors are effective in animal models of disease and have advanced to clinical trials for the treatment of inflammatory diseases and cancer. The clinical utility of specifically targeting a subset of cellular signaling cascades and signaling cascades that regulate pleiotropic cellular processes are being evaluated. The results of these efforts have broad implications for the treatment of many diseases.
Intense efforts are under way to develop potent and selective inhibitors of kinases constituting mitogen-activated protein kinase pathways, which are crucial cell signalling pathways. These efforts have yielded promising compounds that are being evaluated in clinical trials.
WNK (with no lysine K) kinases are serine-threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause ...pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.
An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine ...analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.
Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. ...Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited.
We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC).
Cytometric profiling identified an immunologically "hot" cluster with abundant CD8
T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8
T cells and expression of inhibitory markers. The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10.
Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.
The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.
With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop ...diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.
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