Molecular Response Levels Research Articles

Predicting single-cell cellular responses to perturbations using cycle consistency learning.

Phenotype-based drug screening emerges as a powerful approach for identifying compounds that actively interact with cells. Transcriptional and proteomic profiling of cell lines and individual cells provide insights into the cellular state alterations that occur at the molecular level in response to external perturbations, such as drugs or genetic manipulations. In this paper, we propose cycleCDR, a novel deep learning framework to predict cellular response to external perturbations. We leverage the autoencoder to map the unperturbed cellular states to a latent space, in which we postulate the effects of drug perturbations on cellular states follow a linear additive model. Next, we introduce the cycle consistency constraints to ensure that unperturbed cellular state subjected to drug perturbation in the latent space would produces the perturbed cellular state through the decoder. Conversely, removal of perturbations from the perturbed cellular states can restore the unperturbed cellular state. The cycle consistency constraints and linear modeling in the latent space enable to learn transferable representations of external perturbations, so that our model can generalize well to unseen drugs during training stage. We validate our model on four different types of datasets, including bulk transcriptional responses, bulk proteomic responses, and single-cell transcriptional responses to drug/gene perturbations. The experimental results demonstrate that our model consistently outperforms existing state-of-the-art methods, indicating our method is highly versatile and applicable to a wide range of scenarios. The source code is available at: https://github.com/hliulab/cycleCDR.

Minimal Residual Disease-Negative Complete Remission at the End of Induction Is a Prognostic Indicator of Long-Term Survival in Adult Patients with Ph+ Acute Lymphoblastic Leukemia Receiving First-Line Therapy

Background: Minimal residual disease (MRD) is an established, prognostic indicator for patients with acute lymphoblastic leukemia (ALL), including Philadelphia chromosome-positive (Ph+) ALL. The objective of this study was to evaluate the association between MRD and long-term outcomes (event-free survival [EFS] and overall survival [OS]) in adult patients with Ph+ ALL receiving first-line therapy. Methods: A systematic literature review (SLR) was performed to identify eligible studies, including interventional and observational studies reporting survival outcomes by MRD status in adults ≥18 years of age with Ph+ ALL receiving first-line therapy. Key outcomes and data elements of interest were EFS, OS, and MRD or MRD-negative complete remission (CR) at end of induction (EOI). Aggregate study-level and individual patient data (IPD) meta-analyses were conducted. The IPD approach included a log-level MRD analysis to evaluate the concordance of deepening levels of molecular response with long-term EFS or OS in patients with CR and an analysis evaluating the utility of MRD-negative CR over CR alone. The analyses were conducted using frequentist and Bayesian approaches with fixed-effects and random-effects models. Kaplan-Meier method with log-rank tests was used for survival analysis. Where applicable, Cox regression method with and without adjustment for prognostic factors were applied. Results: The SLR identified 19 publications (18 unique studies), 10 (9 unique studies) of which had sufficient MRD and EFS/OS data. Nine studies (n=704) were included in the aggregate study-level analysis; 3 of the 9 studies (Phase II AP24534-11-001 [Jabbour et al. 2015], Phase II GIMEMAINCB 84344-201 [Martinelli et al. 2017], and Phase III GRAAPH-2005 RCT [Chalandon et al. 2015]) had IPD available. MRD was assessed by polymerase chain reaction in all 9 studies. Findings from the aggregate study-level meta-analysis demonstrated a significant long-term survival benefit (EFS and OS) in favor of patients with MRD-negative CR. This significant benefit was generally consistent across analysis types (base-case or sensitivity analyses), analytic models (fixed- or random-effects), and approaches (frequentist or Bayesian). Evidence from the IPD analysis, based on patients with CR, was mostly consistent with the aggregate study-level meta-analysis, supporting MRD-negative CR at the EOI as being predictive of EFS or OS. Log-level MRD analysis showed that patients who are in CR with deeper molecular response ( BCR::ABL1/ABL1 ≤0.01%) at EOI have significantly better long-term EFS and OS compared with patients with other levels of MRD (ie, BCR::ABL1/ABL1 >0.01%). This significant long-term survival benefit was also demonstrated in patients with MRD-negative CR ( BCR::ABL1/ABL1 ≤0.01%) at EOI compared with patients who were MRD-positive ( BCR::ABL1/ABL1 >0.01%) ( Table 1) or based on achievement of CR alone (regardless of BCR::ABL1/ABL1 values). Results from the IPD analysis standardized for outcome definition, with and without adjustment for prognostic factors (age, gender, white blood cell count at baseline, and stem cell transplant), were consistent with the findings from the log-level MRD analysis. The analysis showed a significant association between MRD-negative/MRD-negative CR status (ie, BCR::ABL1/ABL1 ≤0.01% threshold) at EOI with improved long-term EFS and OS. The results were significant for EFS in both the adjusted and unadjusted models. The OS results were significant only in the unadjusted model. Conclusions: These analyses indicate that a deeper molecular response at EOI results in better long-term EFS and OS; MRD-negative CR has a greater prognostic value than CR and is strongly associated with long-term EFS and OS in patients with Ph+ ALL. Acknowledgement: The authors acknowledge APHP, who provided the individualized patients data for the GRAAPH study.

Цитокиновый статус больных хроническим миелолейкозом

Introduction. Altered cytokine secretion has been described in many types of cancer. Being a key mediator of intercellular interactions, they can contribute to the development of resistance to targeted therapy and cytostatic drugs in chronic myeloid leukemia (CML). Aim. To evaluate the relationship between cytokine spectrum indicators in patients with CML and the effectiveness of targeted tyrosine kinase inhibitors (TKI) therapy. Materials and methods. Quantitative determination of the concentration of cytokines TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-18, IFN-α in blood serum of patients with CML (n = 60) and apparently healthy blood donors (n = 22) was carried out using enzyme-linked immunosorbent assay (ELISA). The phase of the disease and the depth of molecular response were determined according to criteria of the European Leukemia Net 2020. Stratification of CML patients by risk groups was carried out according to the Sokal (1984), EUTOS long-term survival (ELTS) (2016) scores. Hematological and non-hematological toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE v.5.0, 2017). Results. It was found that the serum concentration of a number of cytokines in the group of patients with CML was significantly higher compared to the control group: TNF-α – 2.80 and 0.70 pg/ml, p < 0.001, IL-6 – 3.10 and 0.70 pg/ml, p < 0.001, IL-18 – 296.40 and 122.10 pg/ml, p < 0.001, IL-10 – 5.15 and 0.95 pg/ml, p < 0.001, IFN-α – 5.60 and 4.00 pg/ml, p = 0.006, respectively. Among patients with CML, the level of cytokines varied depending on the level of molecular response and the type of TKI. Patients who did not achieve a major molecular response (MMR) were characterized by a significantly greater increase in the secretion of TNF-α, IL-6 and IL-1β (6.65 and 2.30 pg/ml, p = 0.004, 9.40 and 2.80 pg/ml, p < 0.001, 3.69 and 0.88 pg/ml, p = 0.004, respectively) compared with patients with MMR. However, only IL-6 was a statistically significant prognostic factor influencing the achievement of MMR (odds ratio 1.131, confidence interval 1.015–1.260, p = 0.025). Among patients receiving imatinib therapy, the level of TNF-α was lower compared to patients receiving nilotinib, dasatinib and bozutinib (2.10, 4.00, 6.85 and 4.25 pg/ml, respectively, p = 0.013). Conclusion. The results of the research demonstrated increased secretion of TNF-α, IL-6, IL-18, IL-10, and IFN-α in CML patients, which may indicate an important role in the pathogenesis of CML. TNF-α, IL-6, and IL-1 levels were higher in patients who did not achieve MMR, and IL-6 was a statistically significant predictor the achievement of MMR.

Root and shoot biology of Arabidopsis halleri dissected by WGCNA: an insight into the organ pivotal pathways and genes of an hyperaccumulator.

Arabidopsis halleri is a hyperaccumulating pseudo-metallophyte and an emerging model to explore molecular basis of metal tolerance and hyperaccumulation. In this regard, understanding of interacting genes can be a crucial aspect as these interactions regulate several biological functions at molecular level in response to multiple signals. In this current study, we applied a weighted gene co-expression network analysis (WGCNA) on root and shoot RNA-seq data of A. halleri to predict the related scale-free organ specific co-expression networks, for the first time. A total of 19,653 genes of root and 18,081 genes of shoot were grouped into 14 modules and subjected to GO and KEGG enrichment analysis. "Photosynthesis" and "photosynthesis-antenna proteins" were identified as the most enriched and common pathway to both root and shoot. Whereas "glucosinolate biosynthesis," "autophagy," and "SNARE interactions in vesicular transport" were specific to root, and "circadian rhythm" was found to be enriched only in shoot. Later, hub and bottleneck genes were identified in each module by using cytoHubba plugin based on Cytoscape and scoring the relevance of each gene to the topology of the network. The modules with the most significant differential expression pattern across control and treatment (Cd-Zn treatment) were selected and their hub and bottleneck genes were screened to validate their possible involvement in heavy metal stress. Moreover, we combined the analysis of co-expression modules together with protein-protein interactions (PPIs), confirming some genes as potential candidates in plantheavy metal stress and as biomarkers. The results from this analysis shed the light on the pivotal functions to the hyperaccumulative trait of A. halleri,giving perspective to new paths for future research on this species.

Non-canonical DNA structures: Diversity and disease association.

A complete understanding of DNA double-helical structure discovered by James Watson and Francis Crick in 1953, unveil the importance and significance of DNA. For the last seven decades, this has been a leading light in the course of the development of modern biology and biomedical science. Apart from the predominant B-form, experimental shreds of evidence have revealed the existence of a sequence-dependent structural diversity, unusual non-canonical structures like hairpin, cruciform, Z-DNA, multistranded structures such as DNA triplex, G-quadruplex, i-motif forms, etc. The diversity in the DNA structure depends on various factors such as base sequence, ions, superhelical stress, and ligands. In response to these various factors, the polymorphism of DNA regulates various genes via different processes like replication, transcription, translation, and recombination. However, altered levels of gene expression are associated with many human genetic diseases including neurological disorders and cancer. These non-B-DNA structures are expected to play a key role in determining genetic stability, DNA damage and repair etc. The present review is a modest attempt to summarize the available literature, illustrating the occurrence of non-canonical structures at the molecular level in response to the environment and interaction with ligands and proteins. This would provide an insight to understand the biological functions of these unusual DNA structures and their recognition as potential therapeutic targets for diverse genetic diseases.

Screening and identification of drought tolerance of spring soybean at seedling stage under climate change

Drought is one of the major abiotic stress factors limiting soybean growth and yield, and it frequently occur globally. Therefore, exploring resistant varieties from soybean germplasm is important under climate change. To screen drought resistant spring soybean varieties at seedling stage, pot experiment was used to detect the Survival percentage after drought stress of 60 soybean varieties at seedling stage, twice drought rehydration treatments on seedlings, to evaluate the drought tolerance of spring soybean. The results showed that at the seedling stage, seven varieties were considered drought tolerant, 17 varieties were considered drought sensitive, and 36 varieties were considered to be moderately drought tolerant. Based on this experiment, number 44 (heinong37), 48 (heinong44), 49 (heinong45), 52 (heinong48) is considered the best drought resistant, and number 3 (dongnong48), 4 (dongnong52), 27 (suinong25), 60 (heinong65) is the most sensitive. These varieties provide a reference for further study on drought tolerance and stress resistance gene screening of soybean at the molecular level. The selected soybean varieties can be planted in areas with suitable climates and frequent drought to meet the local soybean demand. In other regions, although cannot be directly grown, they can still be used as parents of selected varieties or as materials for gene screening and extraction, to assist crop breeding at the molecular level in response to increasingly severe drought stress problems under the current climate trends.

NATURAL KILLER CELLS PHENOTYPE CORRELATES WITH TYROSINE KINASE INHIBITORS TREATMENT RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS

Despite well-known Natural Killer (NK) cells antitumor activity, its potential in regulating Chronic Myeloid Leukemia (CML) is not fully stablished. Tyrosine kinase inhibitors (TKI) treatment, besides reducing BCR-ABL1 transcripts, seems to result in immunomodulation that partly restore the immune dysregulation of the disease. Numerical, functional and maturation defects might be associated with CML risk stratification, molecular response profile or even with resistance to treatment with TKI. Therefore, we aimed to immunophenotypically characterize frequency, subtypes and maturation profile of NK cells in CML patients, by Multiparametric Flow Cytometry (MFC), during monitoring of TKI treatment. 11 Peripheral Blood samples from patients with CML (Clinical Hospital of Ribeirão Preto - HCRP-USP) and 10 healthy donors (controls) were analyzed. Clinical, laboratory and ELN2017 risk stratification were assessed. NK cells were defined as CD19-CD3-CD56+ and: CD56brightCD16- (secretory), CD56dimCD16+ (cytotoxic), CD11b-CD27- (tolerant); CD27+CD11b- (immature secretor), CD27+CD11b+ (secretory) and CD11b+CD27- (cytotoxic). Based on molecular response levels assessed by BCR-ABL1 RT-qPCR, patients were classified in G1 (without loss of molecular response to the 1 st line of TKI; n = 3), G2 (loss of molecular response to 1 st line of TKI; n = 3) and G3 (loss of molecular response to 2 nd generation TKI; n = 5). Mann Whitney or Kruskal-Wallis tests (p < 0.05) were applied using SPSS (v.20). A higher frequency of lymphocytes in CML patients was observed when compared to controls (p = 0.0242), but no difference was found in the frequency of NK cells. G1 and G2 showed a higher frequency of NK cells than G3 (p = 0,7774). Even though no statistical significance was found, CD27-CD11b- and CD27+CD11b- NK cells showed a higher frequency, while CD27+CD11b+ NK cells showed a lower frequency in CML when compared to controls. Frequency of CD27+CD11b- NK cells was higher in G2 and G3 when compared to G1. G3 showed an immature profile with higher percentages of CD27-CD11b- and CD27+CD11b- NK cells when compared to controls, while the more mature CD27+CD11b+ NK cells showed a lower frequency in CML. Furthermore, a lower frequency of CD56brightCD16- (p = 0.0002) and CD56dimCD16+ (p = 0.0448) NK cells was observed in CML, as wells as the frequency of CD27-CD11b+ cytotoxic NK cells, consistent to what we found in G3 and supporting our hypothesis. We observed that patients with loss of molecular response in treatment with 1 st and 2 nd TKI generation presented an immature non-effective NK cell profile, with deficient cytotoxicity. This scenario might result in impaired tumor surveillance of CML, meaning that phenotypic changes in NK cells may directly affect risk stratification and treatment response in CML. However due to the small number of patients and the lack of a functional assay, it is not possible to confirm that the cytotoxic cell may actually be deficient. Our findings and further functional studies can contribute to a better understanding of the physiopathology, prognosis and monitoring of CML, response to TKI therapy, as well as help to define therapeutic strategies based on immunotherapy mediated by NK cells.

Transcriptomics Analysis of Heat Stress-Induced Genes in Pepper (Capsicum annuum L.) Seedlings

Pepper (Capsicum annuum L.) is one of the most economically important crops worldwide. Heat stress (HS) can significantly reduce pepper yield and quality. However, changes at a molecular level in response to HS and the subsequent recovery are poorly understood. In this study, 17-03 and H1023 were identified as heat-tolerant and heat-sensitive varieties, respectively. Their leaves’ transcript abundance was quantified using RNA sequencing to elucidate the effect of HS and subsequent recovery on gene expression. A total of 11,633 differentially expressed genes (DEGs) were identified, and the differential expression of 14 randomly selected DEGs was validated using reverse-transcription polymerase chain reaction. Functional enrichment analysis revealed that the most enriched pathways were metabolic processes under stress and photosynthesis and light harvesting during HS and after recovery from HS. The most significantly enriched pathways of 17-03 and H1023 were the same under HS, but differed during recovery. Furthermore, we identified 38 heat shock factors (Hsps), 17 HS transcription factors (Hsfs) and 38 NAC (NAM, ATAF1/2, and CUC2), and 35 WRKY proteins that were responsive to HS or recovery. These findings facilitate a better understanding of the molecular mechanisms underlying HS and recovery in different pepper genotypes.

Transcriptome profiling of Malus sieversii under freezing stress after being cold-acclimated

BackgroundFreezing temperatures are an abiotic stress that has a serious impact on plant growth and development in temperate regions and even threatens plant survival. The wild apple tree (Malus sieversii) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in wild apple trees.ResultsPhytohormone and physiology profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that JA, IAA, and ABA accumulated in the cold acclimation stage and decreased during freezing stress in response to freezing stress. To elucidate the molecular mechanisms of freezing stress after cold acclimation, we employed single molecular real-time (SMRT) and RNA-seq technologies to study genome-wide expression profiles in wild apple. Using the PacBio and Illumina platform, we obtained 20.79G subreads. These reads were assembled into 61,908 transcripts, and 24,716 differentially expressed transcripts were obtained. Among them, 4410 transcripts were differentially expressed during the whole process of freezing stress, and these were examined for enrichment via GO and KEGG analyses. Pathway analysis indicated that “plant hormone signal transduction”, “starch and sucrose metabolism”, “peroxisome” and “photosynthesis” might play a vital role in wild apple responses to freezing stress. Furthermore, the transcription factors DREB1/CBF, MYC2, WRKY70, WRKY71, MYB4 and MYB88 were strongly induced during the whole stress period.ConclusionsOur study presents a global survey of the transcriptome profiles of wild apple trees in dynamic response to freezing stress after two days cold acclimation and provides insights into the molecular mechanisms of freezing adaptation of wild apple plants for the first time. The study also provides valuable information for further research on the antifreezing reaction mechanism and genetic improvement of M. sieversii after cold acclimation.

Mechanical adaptation of brachiopod shells via hydration-induced structural changes

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration.

Altered soil organic matter composition and degradation after a decade of nitrogen fertilization in a temperate agroecosystem

Nitrogen (N) fertilization has been found to alter soil carbon storage in agroecosystem as various soil organic matter (OM) components respond differently to environmental change. However, how the direction and magnitude of soil carbon storage changes in response to N fertilization remains unclear, and a lack of deep understanding on soil OM composition particularly under various fertilization rates constrains our ability to reveal soil OM dynamics. In this study, we investigated soil OM composition and degradation at the molecular-level in response to various rates of N fertilization, and the potential mechanisms that govern these alterations. Soil samples (0−10 cm) were collected from a 10-year N fertilization experiment in Southern Ontario, Canada with increasing rates (non-fertilized control, 30, 87, 145 and 260 kg ha−1 yr−1). The samples were analyzed for soil organic carbon and total N concentrations, soil OM composition (i.e. plant-derived steroids, cutin-, suberin-, lignin-derived compounds), and microbial biomass and community structure. Despite similar soil organic carbon (21.7–23.1 g/kg) and total N contents (2.0–2.2 g/kg) across all treatments, decreased concentrations of plant-derived steroids, cutin- and suberin-derived compounds under some N addition treatments were observed. This was consistent with lower alkyl carbon contents from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy analysis. The lower plant-derived steroids, cutin- and suberin-derived lipids may be explained by the enhanced degradation of steroids and cutin likely associated with higher microbial biomass under N fertilization. Lignin-derived phenols were elevated under N fertilization, which is consistent with the aromatic/phenolic carbon contents (mainly from lignin) from NMR analysis. The enhanced lignin degradation, which is consistent with higher fungal biomass (lignin degraders), did not result in lower lignin-derived compound or aromatic/phenolic carbon content under N addition. As other studies reported higher lignin contents in corn residues under N fertilization, we suggest that the turnover of lignin-derived phenols may be governed not only by degradation processes but also by the quantity and quality of crop residues. Furthermore, we observed that different N rates exert various controls on soil OM cycling. For example, the increase in degradation of steroids, cutin and lignin was smaller above the threshold N rates 145 kg ha−1 yr−1, implying that the degradation of these compounds was fertilization rate-dependent. These results highlight that various N fertilization levels alter the allocation of carbon and soil OM dynamics likely through changing biodegradation and the quantity and quality of crop residues.

Molecular Testing in CML between Old and New Methods: Are We at a Turning Point?

Molecular monitoring of minimal residual disease (MRD) and BCR-ABL1 kinase domain (KD) mutation testing have a well consolidated role in the routine management of chronic myeloid leukemia (CML) patients, as they provide precious information for therapeutic decision-making. Molecular response levels are used to define whether a patient has an “optimal”, “warning”, or “failure” response to tyrosine kinase inhibitor (TKI) therapy. Mutation status may be useful to decide whether TKI therapy should be changed and which alternative TKI (or TKIs) are most likely to be effective. Real-time quantitative polymerase chain reaction (RQ-qPCR) and Sanger sequencing are currently the gold standard for molecular response monitoring and mutation testing, respectively. However, in recent years, novel technologies such as digital PCR (dPCR) and next-generation sequencing (NGS) have been evaluated. Here, we critically describe the main features of these old and novel technologies, provide an overview of the recently published studies assessing the potential clinical value of dPCR and NGS, and discuss how the state of the art might evolve in the next years.

Implications of Abscisic Acid in the Drought Stress Tolerance of Plants

Drought is a severe environmental constraint, which significantly affects plant growth, productivity, and quality. Plants have developed specific mechanisms that perceive the stress signals and respond to external environmental changes via different mitigation strategies. Abscisic acid (ABA), being one of the phytohormones, serves as an important signaling mediator for plants’ adaptive response to a variety of environmental stresses. ABA triggers many physiological processes, including bud dormancy, seed germination, stomatal closure, and transcriptional and post-transcriptional regulation of stress-responsive gene expression. The site of its biosynthesis and action must be clarified to understand the signaling network of ABA. Various studies have documented multiple sites for ABA biosynthesis, their transporter proteins in the plasma membrane, and several components of ABA-dependent signaling pathways, suggesting that the ABA response to external stresses is a complex networking mechanism. Knowing about stress signals and responses will increase our ability to enhance crop stress tolerance through the use of various advanced techniques. This review will elaborate on the ABA biosynthesis, transportation, and signaling pathways at the molecular level in response to drought stress, which will add a new insight for future studies.

Conventional Real Time Quantitative Polymerase Chain Reaction Method Yields Similar Level of Sensitivity to Digital Droplet Polymerase Chain Reaction for Detection of BCR-ABL p210 Transcripts in Patients with Chronic Phase Chronic Myeloid Leukemia

Introduction: Several studies have investigated the utility of digital droplet polymerase chain reaction (ddPCR) in the detection of p210 BCR-ABL fusion transcripts in chronic myeloid leukemia (CML), and have suggested greater sensitivity compared to real-time quantitative PCR (RT-qPCR). There is limited data regarding the utility and sensitivity of ddPCR in patients with chronic phase CML (CP-CML) who are candidates for tyrosine kinase inhibitor (TKI) discontinuation. We aimed to investigate the capability of ddPCR to detect p210 BCR-ABL transcript levels in CP-CML patients and compare the sensitivity to RT-qPCR. Methods: We analyzed 103 different peripheral blood (n=98) and bone marrow (n=5) samples from 36 patients with CP-CML treated at Moffitt Cancer Center between 2013 and 2018. Samples were from patients who either met clinical criteria for TKI discontinuation or were enrolled in a phase I dose-escalation study evaluating combination therapy with nilotinib plus ruxolitinib who had at least a complete cytogenetic response (CCyR) to TKI therapy with detectable BCR-ABL transcripts by RT-PCR at the time of enrollment(1, 2). BCR-ABL levels were assessed by RT-qPCR at the time of collection with level of transcript reported as % International Standard (%IS). Samples collected from 2013 to 2014 were reported as p210 to ABL and converted to %IS per lab-specific conversion factor. Stored purified RNA samples were obtained retrospectively and ddPCR was performed using the QX-200 Droplet Digital PCR System and QXDx BCR-ABL %IS Kit (Bio-Rad). Droplets were analyzed by QXDx BCR-ABL reporter software to determine p210 transcript level (%IS). Analysis for RNA degradation of stored samples was performed via High Sensitivity RNA ScreenTape (Agilent). Samples that did not pass quality check upon performing ddPCR were excluded from further analysis. Correlation coefficient was calculated with p-value of &lt;0.05 being significant. Results: Of the 36 patients, 28 were in the TKI discontinuation cohort and 10 were enrolled in the clinical trial of nilotinib plus ruxolitinib. Two patients who participated in the clinical trial subsequently also met criteria for TKI discontinuation. In the TKI discontinuation cohort, 46% of patients (n=13) lost major molecular response (MMR) with a median time to loss of MMR of 88 days. We observed a statistically significant correlation between RT-qPCR and ddPCR levels when comparing %IS (correlation coefficient R2=0.42; p&lt;0.0001) as well as calculated molecular response (MR) logarithm values of all available samples (R2=0.67; p &lt;0.0001) (Figure 1). There was a weaker correlation between patients determined to have MMR by RT-qPCR (MR≥3.0) compared to ddPCR (R2=0.55; p &lt;0.0001) (Figure 2). A total of 91 different samples were consistent with MMR when tested by RT-PCR. Of these samples, 85.7% (n=78) were consistent with MMR when tested by ddPCR and the remaining 14.3% (n=13) appeared to have higher detectable levels of p210 transcripts (MR&lt;3.0). When investigating deeper molecular responses, 62 samples were consistent with MR≥4.0 by RT-PCR, and 20.9% (n=13) of these samples did not achieve a deep molecular response (MR&lt;4.0) when tested by ddPCR. Of note, 5 representative samples were also evaluated for potential RNA degradation and 3 were noted to have significant degradation present. Conclusion: Compared to RT-PCR, ddPCR represents a viable method for detection of p210 transcripts for patients with CP-CML. The two methods yield similar efficacy when detecting varying levels of molecular response, however further research is warranted for the ability to detect deeper molecular responses for patients in MMR. Our study is limited by inherent retrospective bias and quality of RNA in stored samples. 1. National Comprehensive Cancer Network. Chronic Myeloid Leukemia (Version 1.2019). 2. Sweet K, Hazlehurst L, Sahakian E, Powers J, Nodzon L, Kayali F, et al. A phase I clinical trial of ruxolitinib in combination with nilotinib in chronic myeloid leukemia patients with molecular evidence of disease. Leuk Res. 2018;74:89-96. Disclosures Talati: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Honoraria; Agios: Honoraria. Shelton:Bio-Rad: Employment. Nodzon:Pharmacyclics: Consultancy; Abbvie: Other: Speaker Fees; Genentech: Consultancy, Other: Speaker Fees; Pfizer: Consultancy. Sweet:Stemline: Consultancy; Pfizer: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Jazz: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees. Pinilla Ibarz:Novartis: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; TG Therapeutics: Consultancy; Teva: Consultancy; Sanofi: Speakers Bureau.

Imatinib in Children with Chronic Myeloid Leukemia in Chronic Phase: Long-Term Results of the French National Phase IV Trial

Because of the rarity of Chronic Myeloid Leukemia (CML) in children and adolescents, only few studies reported on efficacity and tolerance of imatinib in the pediatric population and scant data are available regarding long-term follow-up. The aim of our analysis was to assess, the long-term efficacity and safety of imatinib in children with CML in early chronic phase included in the French multicentric prospective Glivec Phase IV trial (Millot et al, J Clin Oncol 2011). Methods: Children aged 0 to 18 years with newly diagnosis CML in chronic phase were eligible to received daily imatinib 260 mg/m² according the trial. Long-term analysis included overall survival (OS), progression-free survival (PFS), response to treatment and adverse events. Results: Between March 2004 and December 2008, 44 patients (median age 13.4 years; range 0.8 - 16.7 years) were included in the trial. As of April 2019, with a median follow-up of 10.6 years (range 1.8 - 13.4 years), 2 patients (pts) progressed to blastic crisis and only one death was recorded. The median age was 21.8 years (range 9.3 - 28.8 years) at the last follow-up. The median duration of imatinib therapy was 10.5 years (range 0.2 - 12.5 years) for the entire cohort. To date, 13 pts (29.5%) are still treated with imatinib. Thirty-one pts (70.5%) had discontinued first line treatment with imatinib after a median time of treatment of 2.4 years (range 0.2 - 10.6 years) for the following reasons: 10 pts did not achieve major molecular response (MMR), 1 pt developed blast crisis, 2 pts had unsatisfactory level of molecular response (MR) according to the clinician, 10 pts lost their response (loss of complete hematological response n=1, complete cytogenetic response [CCR] n=6 and MMR n=3), 4 pts attempted treatment free remission (TFR), 3 pts were intolerant to imatinib and 1 pt stopped because of pregnancy. Among these 31 pts who discontinued imatinib, 2 pts are still in TFR, and 29 pts switched to a second line therapy: second generation tyrosine kinase inhibitors (2TKI) (n=25), allogeneic hematopoietic stem cell transplantation (HSCT) (n=3), polychemotherapy (n=1). Sixteen of these 31 pts (51.6%) required subsequent lines of therapy including a second pt who transformed to blastic crisis under a second line therapy with dasatinib. Overall 11 pts (25%) underwent HSCT. Overall, regarding the best response, during the study follow-up 11 pts (25%) achieved MMR after a median time of 2.3 years (range 0.8-5.1), 7 pts (13.6%) achieved MR4 after a median time of 5.1 years (range 2.5-7.8), 25 pts (56.8%) achieved MR4.5 after a median time of 2.92 years (range 1.1-10.4) and 1 pt (2.3%) achieved CCR only. At last follow-up, 43 out the 44 pts were alive : 3 pts (7%) were in CCR, 12 pts (27.9%) in MMR, 6 pts (13.9%) in MR4 and 22 pts (51.2%) in MR4.5. Among the 13 pts still treated with imatinib, 1 pt (7.7%) was in CCR, 6 pts (14%) in MMR, 3 pts (23.1%) were in MR4 and 3 pts (23.1%) in MR4.5. Among the 11 transplanted patients, all pts except one are alive, in at least MR4.5. The death was related to post transplant infection. On an intention to treat basis, the 10-year OS of 44 patients treated was 97.7% (CI 95% 93.3-100). The 10-year PFS was 95.5% (CI 95% 89.3-100). We collected also the long-term safety of imatinib in the 25 pts who have received this therapy for more than 4 years. Newly occurring or worsening grade 3 or 4 hematologic or biochemical adverse events were infrequent after 4 years of imatinib. There is a decrease in the frequency of hematologic and extra hematologic sides effects reported during the first year and those reported after the fourth year of treatment with imatinib: musculoskeletal events 80 vs 24% (p&lt;0,0001), abdominal pain 44% vs 16% (p=0,03), nausea 48% vs 16% (p=0,02), diarrhea 24% vs 0% (p=0,01) and neutropenia 84% vs 28% (p&lt;0,0001), respectively. Conversely, the incidence of lymphopenia appeared with duration of imatinib treatment (p=0,04). Conclusion: With more than 10 years of follow-up, we showed that imatinib remains effective in one third of children included in the Glivec phase IV study with acceptable adverse effects and a low impact over time. Despite the notable proportion of switches, the OS and the PFS remain satisfactory in this pediatric cohort. Disclosures No relevant conflicts of interest to declare.

PF403 NEGATIVE MR4.0 CHRONIC MYELOID LEUKEMIA AND ITS IMPLICATIONS FOR TREATMENT‐FREE REMISSION

Background:In the last few years, several clinical discontinuation trials have demonstrated that 40–60% of chronic phase CML patients (CP‐CML) who have achieved a stable deep molecular response, can stop therapy without relapsing. However, there is no consensus as to whether or not the depth of the molecular response is a prognostic factor for treatment‐free remission (TFR), which is reflected in the discrepancy between the various guidelines and opinions of experts in the field regarding the optimal molecular response required, MR4.0 or MR4.5, for a TFR attempt.Aims:In this work, we hypothesize that a discrepancy in the classification of the level of molecular response between laboratories involved in clinical trials could introduce a bias in the evaluation of the prognostic impact of treatment‐free remission (TFR).Methods:In an attempt to clarify this issue, we stratified by molecular response a cohort of 1087 CML patients and compared our results with those of 200 patients enrolled in the EURO‐SKI trial.Results:We found that none of the patients classified as MR4.0 had detectable disease (0/169) (Figure 1A). In addition, the frequency of undetectable disease negative cases increased to 29% (61/211) and 65% (149/230) in the MR4.5 and MR5.0 groups, respectively (Figure 1A). By contrast, the frequency of MR4.0 with undetectable disease in the EURO‐SKI trial was 72% (49/68), decreasing to 56% (48/85) and 58% (25/43) in the MR4.5 and MR5.0 groups, respectively (Figure 1B).Summary/Conclusion:Our results suggest that (1) There may be a bias in patient stratification by molecular response in the EURO‐SKI trial, making it impractical to attempt any association between molecular response depth and TFR success, (2) we should reassess the current classification of MR4.0 to include only patients with detectable disease and (3) the minimum quality criteria for scoring molecular response when disease is undetectable should be raised, with samples with &lt;32000 ABL1 or &lt;77000 GUSB transcripts be considered as unevaluable for classification of DMR.image

De novo transcriptomic profiling of the clonal Leymus chinensis response to long-term overgrazing-induced memory

Sheepgrass (Leymus chinensis) is one of the dominant grass species present on typical steppes of the Inner Mongolia Plateau. However, L. chinensis has developed a dwarfing phenotype in response to the stressful habitat in grasslands that are severely degraded due to heavy grazing. The lack of transcriptomic and genomic information has prohibited the understanding of the transgenerational effect on physiological alterations in clonal L. chinensis at the molecular level in response to livestock grazing. To solve this problem, transcriptomic information from the leaves of clonal L. chinensis obtained from overgrazed (GR) and non-grazed (NG) grasslands was studied using a paired-end Illumina HiSeq 2500 sequencing platform. First, despite the influence of grazing being absent during the growth of clonal offspring in our hydroponic experiment, compared with those from the NG group, clonal L. chinensis from the GR group exhibited significant dwarf-type morphological traits. A total of 116,356 unigenes were subsequently generated and assembled de novo, of which 55,541 could be annotated to homologous matches in the NCBI non-redundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), gene ontology (GO), or Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The expression of 3,341 unigenes significantly differed between the GR group and the NG group with an absolute value of Log2 ratio ≥ 1. The altered expression of genes involved in defence and immune responses, pathogenic resistance and cell development indicates that livestock grazing induces a transgenerational effect on the growth inhibition of clonal L. chinensis. The results of the present study will provide important large-scale transcriptomic information on L. chinensis. Furthermore, the results facilitated our investigation of grazing-induced transgenerational effects on both the morphological and physiological characteristics of L. chinensis at the molecular levels.

Molecular monitoring of patients with ETV6-PDGFRB rearrangement: Implications for therapeutic adaptation.

Molecular monitoring of patients with ETV6-PDGFRB rearrangement: Implications for therapeutic adaptation.

Immune Effector Recovery in Chronic Myeloid Leukemia and Treatment-Free Remission.

Chronic myeloid leukemia (CML) is a hematological cancer, characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 [t(9;22)], producing the Bcr-Abl oncogene. Tyrosine kinase inhibitors (TKIs) represent the standard of care for CML patients and exert a dual mode of action: direct oncokinase inhibition and restoration of effector-mediated immune surveillance, which is rendered dysfunctional in CML patients at diagnosis, prior to TKI therapy. TKIs such as imatinib, and more potent second-generation nilotinib and dasatinib induce a high rate of deep molecular response (DMR, BCR-ABL1 ≤ 0.01%) in CML patients. As a result, the more recent goal of therapy in CML treatment is to induce a durable DMR as a prelude to successful treatment-free remission (TFR), which occurs in approximately half of all CML patients who cease TKI therapy. The lack of overt relapse in such patients has been attributed to immunological control of CML. In this review, we discuss an immunological timeline to successful TFR, focusing on the immunology of CML during TKI treatment; an initial period of immune suppression, limiting antitumor immune effector responses in newly diagnosed CML patients, linked to an expansion of immature myeloid-derived suppressor cells and regulatory T cells and aberrant expression of immune checkpoint signaling pathways, including programmed death-1/programmed death ligand-1. Commencement of TKI treatment is associated with immune system re-activation and restoration of effector-mediated [natural killer (NK) cell and T cell] immune surveillance in CML patients, albeit with differing frequencies in concert with differing levels of molecular response achieved on TKI. DMR is associated with maximal restoration of immune recovery in CML patients on TKI. Current data suggest a net balance between both the effector and suppressor arms of the immune system, at a minimum involving mature, cytotoxic CD56dim NK cells may be important in mediating TFR success. However, a major goal remains in CML to identify the most effective pathways to target to maximize an advantageous immune response and promote TFR success.

Direct high-pressure NMR observation of dipicolinic acid leaking from bacterial spore: A crucial step for thermal inactivation

A bacterial spore protects itself with an unusually high concentration (~10% in dry weight of spore) of dipicolinic acid (DPA), the release of which is considered the crucial step for inactivating it under mild pressure and temperature conditions. However, the process of how the spore releases DPA in response to pressure remains obscure. Here we apply 1H high-resolution high-pressure NMR spectroscopy, for the first time, to the spore suspension of Bacillus subtilis natto and monitor directly and in real-time the leaking process of DPA in response to pressure of 200MPa at 20°C. We find that about one third of the total DPA leaks immediately upon applying pressure, but that the rest leaks slowly in hrs upon decreasing the pressure. Once DPA is fully released from the spore, the proteins of the spore become easily denatured at a mild temperature, e.g., 80°C, much below the temperature commonly used to inactivate spores (121°C). The success of the present experiment opens a new avenue for studying bacterial spores and cells at the molecular level in response to pressure, temperature and other perturbations.