Sensitive Mutant Research Articles

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

A Real-world Study on the Expression Characteristics of PD-L1 in Patients with Advanced EGFR Positive NSCLC and Its Relationship with the Therapeutic Efficacy of EGFR-TKIs

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is the first choice for first-line treatment of advanced patients with EGFR positive non-small cell lung cancer (NSCLC). For advanced NSCLC patients with negative drive gene and positive programmed cell death ligand 1 (PD-L1) or highly expressed NSCLC patients, the first-line treatment recommends immune checkpoint inhibitors (ICIs) monotherapy or ICIs combined chemotherapy. Therefore, the first-line treatment strategy for advanced NSCLC patients with EGFR positive at different PD-L1 expression levels is worth further exploring. Many previous studies have suggested that the expression of PD-L1 is obviously affected by EGFR mutation, and the expression of PD-L1 is likely to be related to the mechanism of EGFR-TKIs resistance. The purpose of this study was to analyze the expression characteristics of PD-L1 in patients with advanced EGFR positive NSCLC and its relationship with the efficacy of EGFR-TKIs. 159 patients with newly diagnosed advanced NSCLC with EGFR positive (including 141 patients with EGFR sensitive mutations) were enrolled to analyze the relationship between clinicopathological characteristics and PD-L1 expression. The factors affecting the therapeutic effect of EGFR-TKIs in 141 patients with EGFR sensitive mutations were also explored. The PD-L1 expression of the included patients was classified according to the tumor promotion score (TPS): negative (TPS<1%) accounted for 47.2%, low expression (1%≤TPS<50%) accounted for 32.1%, and high expression (TPS≥50%) accounted for 20.7%. Among them, patients with solid predominant cancer cell pathomorphology classification are more likely to have high expression of PD-L1, accounting for 52.9% (P<0.0001). The median progression-free survival (mPFS) of patients with PD-L1 negative expression, low expression and high expression who received first generation EGFR-TKIs treatment were 12.4 months, 10.5 months and 3.7 months, respectively, with significant statistical difference (P<0.0001). During the EGFR-TKIs treatment, patients with a history of radiotherapy for lung lesions have a longer PFS benefit than those without a history of radiotherapy (mPFS: 17.0 months vs 9.3 months, P<0.0001). The expression level of PD-L1 in advanced EGFR positive NSCLC patients was significantly correlated with the pathomorphology classification of cancer cells. The efficacy of EGFR-TKIs in patients with PD-L1 overexpression NSCLC was significantly poor. PFS can be significantly prolonged during targeted therapy combined with radiotherapy of lung lesions.

A magnesium transporter is involved in the cesium ion resistance of the high-concentration cesium ion-resistant bacterium Microbacterium sp. TS-1.

Cesium ion (Cs+) resistance has been reported in bacteria but is poorly understood as reports on Cs+-resistant bacteria have been limited. We previously reported a novel Cs+/H+ antiporter CshA implicated in Cs+-resistance in Microbacterium sp. TS-1. The present study used the same screening method to isolate novel Cs+-sensitive mutants and their revertants from TS-1. A comparative mutation site analysis using whole-genome sequencing revealed that MTS1_03028 encodes the Mg2+ transporter MgtE and is a candidate Cs+ resistance-related gene. We performed a bioinformatic analysis of MTS1_03028 and complementation experiments on Cs+ resistance in the TS-1 MTS1_03028 mutants Mut5 and Mut7 as well as Escherichia coli expressing MTS1_03028 in the presence of Mg2+. We established the role of MgtE in Cs+ resistance through a functional analysis of TS-1. Enhancing Mg2+ transport by expression of MTS_03028 conferred increased Cs+ resistance. When this strain was exposed to Cs+ concentrations exceeding 200 mM, CshA consistently lowered the intracellular Cs+ concentration. To our knowledge, the present study is the first to clarify the mechanism of Cs+ resistance in certain bacteria. The study findings offer important insights into the mechanism of bacterial resistance to excess Cs+ in the environment, suggesting the potential for bioremediation in high Cs-contaminated areas.

A universal and sensitive gene mutation detection method based on CRISPR-Cas12a

Single nucleotide mutations are highly related to the occurrence and development of cancer. The development of simple single nucleotide mutation detection methods with high sensitivity and specificity has great clinical significance for the prevention, diagnosis, treatment and prognosis evaluation of cancer. In recent years, CRISPR/Cas12a has been developed as a highly sensitive, simple and fast tool for nucleic acid detection. However, the specificity and universality of current detection methods based on it are still insufficient, so their clinical applications are limited. Herein, we developed a simple and rapid single nucleotide mutation detection method based on CRISPR/Cas12a system. This method not only solves the problem of PAM sequence restriction of CRISPR/Cas12a, but also significantly improves the specificity of CRISPR/Cas12a for single nucleotide mutation and greatly improves the sensitivity. We detected three clinically significant mutations, PTEN R130Q, BRAF V600E, and TP53 R248W, with a detection limit of 0.1%. Finally, we further verified the clinical practicability of this method. We selected TP53 R248W mutation site for testing. The accuracy of testing results for 10 clinical samples was as high as 100%. In conclusion, the detection method of specific PCR combined with CRISPR/Cas12a is simple, rapid, universal and highly sensitive. We believe that this method has promising application prospects in clinical diagnosis of cancer.

Gossypol acetate: A natural polyphenol derivative with antimicrobial activities against the essential cell division protein FtsZ.

Antimicrobial resistance has attracted worldwide attention and remains an urgent issue to resolve. Discovery of novel compounds is regarded as one way to circumvent the development of resistance and increase the available treatment options. Gossypol is a natural polyphenolic aldehyde, and it has attracted increasing attention as a possible antibacterial drug. In this paper, we studied the antimicrobial properties (minimum inhibitory concentrations) of gossypol acetate against both Gram-positive and Gram-negative bacteria strains and dig up targets of gossypol acetate using in vitro assays, including studying its effects on functions (GTPase activity and polymerization) of Filamenting temperature sensitive mutant Z (FtsZ) and its interactions with FtsZ using isothermal titration calorimetry (ITC), and in vivo assays, including visualization of cell morphologies and proteins localizations using a microscope. Lastly, Bacterial membrane permeability changes were studied, and the cytotoxicity of gossypol acetate was determined. We also estimated the interactions of gossypol acetate with the promising target. We found that gossypol acetate can inhibit the growth of Gram-positive bacteria such as the model organism Bacillus subtilis and the pathogen Staphylococcus aureus [both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA)]. In addition, gossypol acetate can also inhibit the growth of Gram-negative bacteria when the outer membrane is permeabilized by Polymyxin B nonapeptide (PMBN). Using a cell biological approach, we show that gossypol acetate affects cell division in bacteria by interfering with the assembly of the cell division FtsZ ring. Biochemical analysis shows that the GTPase activity of FtsZ was inhibited and polymerization of FtsZ was enhanced in vitro, consistent with the block to cell division in the bacteria tested. The binding mode of gossypol acetate in FtsZ was modeled using molecular docking and provides an understanding of the compound mode of action. The results point to gossypol (S2303) as a promising antimicrobial compound that inhibits cell division by affecting FtsZ polymerization and has potential to be developed into an effective antimicrobial drug by chemical modification to minimize its cytotoxic effects in eukaryotic cells that were identified in this work.

Fungicide sensitivity levels in the Lithuanian Zymoseptoria tritici population in 2021.

Zymoseptoria tritici causes the disease known as septoria leaf blotch in winter wheat and is a major factor in yield loss worldwide. Farmers are inclined to use fungicides to protect their crops; however, the efficacy of these measures is rapidly decreasing due to the natural mechanisms of mutation emergence in pathogen populations. Increasing fungicide resistance is being recorded worldwide, therefore, screening of the current situation in Lithuania is essential to determine the subsequent steps of crop protection strategies. In this study, in vitro fungicide sensitivity tests, mutation detection, and field experiments were carried out. The mean EC50 values for prothioconazole-desthio and mefentrifluconazole were 0.14 and 0.28 mg/l, respectively. Increased frequency of the mutation S524T, linked to DMIs resistance, was observed. Results revealed that the dominant point mutation in the gene CYP51 was I381V, and the most frequent CYP51 haplotype was D13 (V136C, I381V, Y461H, S524T). The mutation G143A, linked to QoI resistance, was detected in ¾ of the population. Mutations conferring resistance to SDHIs were not detected in single pycnidium isolates. Two-year field experiments likewise showed no decline in field efficacy of SDHI fungicide in Lithuania. Moreover, the baseline sensitivity of the Lithuanian Z. tritici population to QiI fungicide fenpicoxamid was established. The findings of this study provide an update on the current status of fungicide resistance in the Lithuanian Z. tritici population.

Comparative gene analysis of beer tolerant and sensitive Lactobacillus brevis

Lactobacillus brevis is the major spoiler in beer, affecting product quality and causing economic losses in breweries. Although several genes involved in beer-spoilage have been identified, there are still unresolved questions about which genes that are associated with growth in beer and the potential roles of these genes. In this study, 21 Lactobacillus brevis strains were tested for beer spoilage potential; three beer-tolerant and three beer-sensitive strains were selected for comparative genomic analysis. One of the tolerant strains was exposed to growth conditions containing novobiocin that favor loss of plasmids, and subsequently became more sensitive to beer as determined by growth experiments. The genetic difference between the wildtype and the sensitive mutant confirmed that the previously identified beer (hop)-tolerance genes horA and hitA, which are usually localized on plasmids, play important roles in beer spoilage. Interestingly, horA and hitA were present in another sensitive strain whereas these genes were absent in another tolerant strain. This indicates that a beer-spoilage phenotype cannot be easily identified from the presence of a few reported beer-spoilage genes. The potential roles of additional genes involved in beer tolerance were discussed, including a ClpX protease and a manganese transporter different from hitA.

Filamentous temperature sensitive mutant Z: a putative target to combat antibacterial resistance.

In the pre-antibiotic era, common bacterial infections accounted for high mortality and morbidity. Moreover, the discovery of penicillin in 1928 marked the beginning of an antibiotic revolution, and this antibiotic era witnessed the discovery of many novel antibiotics, a golden era. However, the misuse or overuse of these antibiotics, natural resistance that existed even before the antibiotics were discovered, genetic variations in bacteria, natural selection, and acquisition of resistance from one species to another consistently increased the resistance to the existing antibacterial targets. Antibacterial resistance (ABR) is now becoming an ever-increasing concern jeopardizing global health. Henceforth, there is an urgent unmet need to discover novel compounds to combat ABR, which act through untapped pathways/mechanisms. Filamentous Temperature Sensitive mutant Z (FtsZ) is one such unique target, a tubulin homolog involved in developing a cytoskeletal framework for the cytokinetic ring. Additionally, its pivotal role in bacterial cell division and the lack of homologous structural protein in mammals makes it a potential antibacterial target for developing novel molecules. Approximately 2176 X-crystal structures of FtsZ were available, which initiated the research efforts to develop novel antibacterial agents. The literature has reported several natural, semisynthetic, peptides, and synthetic molecules as FtsZ inhibitors. This review provides valuable insights into the basic crystal structure of FtsZ, its inhibitors, and their inhibitory activities. This review also describes the available in vitro detection and quantification methods of FtsZ-drug complexes and the various approaches for determining drugs targeting FtsZ polymerization.

Abstract P049: A simplified, potentially point-of-care (POC), electrode method detects changes in the amount of cfDNA/ctDNA and evaluates the response of advanced cancer patients to therapy

Abstract Rapid assessment of therapy for advanced cancer, especially immunotherapy (IO), is critical. For advanced cancers most therapies, even targeted/personalized ones fail. Even worse, IO has been reported to cause more than doubling in the cancer growth rate (defined as hyper-progressive disease or HPD) in 5-40% (typically ~20%) of patients. Early identification of HPD, or any inactive therapy, and switching to an effective therapy can be lifesaving. Though a consensus has not emerged, there are several reports that describe sensitization to chemotherapy following failed immunotherapy, this suggests that, at least in some cases, switching results in dramatic benefit. Due to inflammatory infiltrates causing “pseudoprogression” (false readings of cancer progression) early during treatment with IO, ineffective IO is often continued for 4-6 months before imaging can definitively say the IO is failing. During this time progressive, potentially fatal cancer growth, as well as medical and financial side effects can occur. The cost of this ineffective or counterproductive IO is typically $10,000 to over $20,000 a month. There are numerous reports of highly sensitive mutation based, or methylation sequence based, cfDNA signatures and methods. Unfortunately, they are inherently difficult tests which typically cost thousands of dollars and do not report the results to clinicians for 1 to 2 weeks. They have not been adopted for routine use to monitor on every visit basis and allow near real-time adjustment of therapy. Rapid detection of failure can lead to lifesaving therapy changes; detection of benefit can enhance compliance and improve clinical outcomes. Previously a research lab style solid gold electrode, requiring artful polishing and reuse, has been reported to be selectively sensitive to cancer DNA due to ctDNA’s altered pattern of methylation. Electrochemical detection of the binding of cfDNA, following a 10-minute incubation, has been published. Details of the dramatically revised and optimized binding methodology are in the methods section of this poster. Compared to previous methods an over 2-fold increase in cfDNA binding is shown. Cancer samples not distinguishable from normal with the previous method show statistically significant detection with the new method. Dramatically increased ability to separate cancer from normal DNA is shown. The new method uses inexpensive, clinically and POC suitable, disposable electrodes; it is highly sensitive to clinical samples. 5 µL of a 10 pg/ul sample provides reproducible readings. cfDNA from normal persons generates readings significantly different than from those with cancer (p &amp;lt; .01). Patient time courses illustrate the correlation of the electrode response with cancer burden and suggests this is a monitoring tool that should be compared to, and may have benefits over, previously approved monitoring tests for advanced cancer such as carcinoembryonic antigen (CEA). Citation Format: Floyd Taub, Matt Russo, Shannon Tunney, Suzin Wright, Chuck Henry. A simplified, potentially point-of-care (POC), electrode method detects changes in the amount of cfDNA/ctDNA and evaluates the response of advanced cancer patients to therapy. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P049.

CircSETD3 mediates acquired resistance to gefitinib in non-small lung cancer cells by FXR1/ECT2 pathway.

Gefitinib is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR sensitive mutation. However, acquired resistance significantly limits its therapeutic efficacy. CircSETD3 has been reported to promote gefitinib resistance in NSCLC cells, however, its underlying mechanisms have not been fully clarified. The expression of circSETD3 were detected in NSCLC patients who received gefitinib as first-line treatment, including 20 gefitinib-sensitive patients and 20 acquired gefitinib-resistant patients. Cell viability were examined by CCK8 assay. The mRNA and protein levels were detected by qRT-PCR and western blot. Using RNA pull-down assay followed by mass spectrometry to identified proteins that interact with circSETD3. The interaction between circSETD3 and fragile X-related protein-1 (FXR1) were further validated by RNA immunoprecipitation (RIP) and pull-down analysis. Fuorescence in situ hybridization (FISH) and immunofluorescence (IF) assays was used for the identification of sub-location of circSETD3 and FXR1 in cells. The effect of circSETD3 overexpression and knockdown on NSCLC tumor growth to gefitinib sensitivity was detected using the mouse xenograft model. CircSETD3 was significantly upregulated in gefitinib-resistant NSCLC cells, and decreased the gefitinib sensitivity in vitro and in vivo. Mechanically, circSETD3 facilitated FXR1 binding to its downstream mRNA target, epithelial cell-transforming sequence 2 (ECT2), promoting ECT2 mRNA decay, which further inhibited cellular apoptosis. CircSETD3/FXR1/ECT2 axis plays a critical role in the acquired resistance to gefitinib in NSCLC. Our results highlight the potential of circSETD3 as a biomarker and therapeutic target for NSCLC patients with acquired gefitinib resistance.

MaHAK5, a Potassium Transporter of Banana, Enhanced Potassium Uptake in Transgenic Arabidopsis under Low Potassium Conditions

Potassium (K+) is one of the most important macronutrients for plant growth and development. It is generally accepted that the KUP/HAK/KT transporters play essential roles in K+ uptake at low concentrations. However, their physiological functions in bananas remain unknown. Here, we cloned MaHAK5 and analyzed its functions in banana (Musa acuminata). Gene expression analysis showed that MaHAK5 was upregulated in the roots and leaves in the early stage of low K+ (LK) stress. MaHAK5 was localized in the cytomembrane. The expression of MaHAK5 improved the growth of the low K+-sensitive yeast mutant R5421 at different K+ supply levels. Overexpression of MaHAK5 in Arabidopsis thaliana significantly enhanced the ability for K+ uptake and increased the chlorophyll content under LK stress. These results indicate that MaHAK5 plays a crucial role in maintaining K+ uptake in bananas.

THERMAL TOLERANCE PHENOTYPE IN CDC13-1 EXO1 HETEROZYGOUS DIPLOIDS OF S. CEREVISIAE IS A DOMINANT TRAIT

Background: Telomeric DNA is found at the end of eukaryotic chromosomes, where they play a role in protecting the chromosome and the integrity of the genome of the organism through the activity of telomerase. Saccharomyces cerevisiae exists in two genotypes: haploid and diploid. Temperature sensitive point mutation on the cdc13 gene of each genotype and deletion of exo1 gene (cdc13-1Exo1 mutants) give rise to mutant survivors at enhanced temperatures. The mode of inheritance of the thermal tolerance allele in the heterozygous diploid genotype is not known. Materials and Methods: We constructed diploids by mating temperature sensitive haploid strains of opposite mating type cdc13-1 exo1: LEU with temperature resistant strains of cdc13-1 exo1::HIS. The crosses were 1296x3182 (D) and 2561x3181 (C). Using a sterile stick, smear of one haploid strain was made on each YEPD plates labelled C2, C8, C10, D4, D10, and D113. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate for six hours, before culturing on media lacking Luecine and Histidine (–L and –H) to purify and confirm that they are diploids. After confirmation, a loop full aliquot of the diploids were streaked on sterile media lacking leucine and histidine (-L, -H) and on YEPD and cultured at 370C to check thermal tolerance and number of viable colonies from each diploid crosses in (cfu). Result: The heterozygous diploid D thrived at the enhanced temperature of 370C and there is a significant difference in the yield of viable colonies by the D diploids when compared to the yield of the C diploids with P-value of 0.05. Conclusion: The growth of diploid D10 as shown in plate 3.1 establishes that, temperature resistant allele inherited by cdc13-1 exo1heterozygous diploids is a dominant phenotype, and its mode of inheritance is dominant as the heterozygous diploid thrived at the enhanced temperature of 370C.

Rationale and protocol design of a phase II study of first-line osimertinib treatment for patients with poor performance status and EGFR mutation-positive non-small cell lung cancer (OPEN/TORG2040)

BackgroundCancer chemotherapy indications for patients with poor performance status and advanced lung cancer are limited. Molecular targeted drugs, including epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, can be used in patients with poor performance status owing to their high efficacy and safety. The third-generation EGFR-tyrosine kinase inhibitor osimertinib has demonstrated effectiveness in the initial treatment of advanced EGFR mutation-positive non-small cell lung cancer in patients with good performance status; however, no evidence exists of the drug’s effectiveness in patients with poor performance status in a prospective study. We designed a study that aims to investigate the efficacy and safety of first-line osimertinib treatment in patients with advanced non-small cell lung cancer harboring sensitive EGFR mutations and with poor performance status.MethodsThe OPEN/TORG2040 study is a multicenter, single-arm, phase II trial for patients with unresectable, advanced EGFR mutation-positive non-small cell lung cancer with a poor performance status (≥ 2). Eligible patients will receive osimertinib until disease progression or unacceptable toxicity. The primary endpoint is the objective response rate of the first-line osimertinib treatment. Considering a threshold value of 45%, expected value of 70% for objective response rate, one-sided significance level of 5%, statistical power of 80%, and ineligible patients, the sample size was set to 30. The secondary endpoints are disease control rate, performance status improvement rate, and safety and patient-reported outcomes using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core Quality of Life Questionnaire and Lung Cancer 13. Time to treatment failure, progression-free survival, and overall survival will also be assessed.DiscussionOur study can determine the clinical benefits of osimertinib treatment in patients with poor performance status, since the clinical outcomes of patients with EGFR mutation-positive non-small cell lung cancer with poor performance status treated with this drug as a first-line treatment have not been sufficiently evaluated.Trial registrationJapan Registry of Clinical Trials: jRCTs041200100 (registration date: February 12, 2021).

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/μL in a linear range of 6.01 × 105 to 3.76 × 108 copies/μL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

Zoledronic acid-associated osteonecro sis of the jaw in patients with EGFR-sensitive mutant lung cancer: two case reports.

Bone metastasis from lung cancer predicts a decrease in the quality of life and a shortening of survival for patients. While controlling the primary disease, active prevention and treatment of skeletal-related events (SREs) are crucial. The use of bisphosphonates as a basic drug for bone metastases from lung cancer has been increasing; also, the corresponding adverse effects have emerged. The case is here reported of two cases of osteonecrosis of the jaw associated with zoledronic acid treatment were reported in patients with epidermal growth factor receptor -sensitive mutation non-small-cell lung cancer(NSCLC) and discussed the clinical features, early recognition and interventions.

In Vitro Evidence of Double Blockade of Asciminib with Reduced Dose of ATP-Binding Pocket Inhibitors in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation

Background Asciminib (ASC), a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, inhibiting BCR-ABL1 via allosteric binding. ASCEMBL trial showed superior efficacy of ASC to Bosutinib (BOS) with a higher molecular response rate, and a lower rate of discontinuation due to lack of efficacy (24.2% vs 35.5% at week 96). However, 24.2% of the patients still experienced lack of efficacy with ASC monotherapy. Also, out of the 39 patients who discontinued ASC due to lack of efficacy, 22 patients (56%) showed a new or persistent ABL1 kinase domain (KD) mutation either on ATP binding sites or Myristoyl pocket site. This finding suggests that ASC monotherapy is not always sufficient to overcome TKI-resistance particularly in those who already experienced multiple TKI failure. Accordingly, dual inhibition of ABL1 kinase protein with ASC combined with ATP binding pocket inhibiter (ABPI) is very attractive option to overcome TKI resistance. The remaining question is dose. A phase 1 study attempted to determine appropriate dose of ASC in combination with fixed dose of ABPIs including Imatinib (IMA), Dasatinib (DAS) and Nilotinib (NIL). But, this approach with fixed dose of ABPIs could add up toxicities not just from ABPIs but also from the combination of two drugs. Accordingly, a different strategy is to be developed to reduce the exposure to ABPI, while combining with ASC. In the present study, we simulated the combination treatment of ASC with ABPIs based on a clinically accessible fixed-dose for various ABL1 mutations to obtain answers to the following questions:Can combination of ASC with 4 different type of ABPIs be universally effective to suppress CML cell growth via dual blockade?Is there a specific ABPI for dual blockade in a specific ABL1 kinase domain mutation?What is the drug concentration of ABPIs for dual blockade when combined with fixed dose of ASC? Methods and Materials In order to simulate the combination treatment of ASC with ABPIs, the IC50 for ASC and ABPI measured from wild type K562 and BaF3BCR-ABL+cell lines were applied: ASC (3nM), IMA (150nM), BOS (10nM), DAS (0.8nM) and NIL (10nM) was applied in the wild type (WT) BaF3 cell lines and cell inhibition was assessed with WST-8 assay. 8 different BaF3 cell lines including wild type, G250E, Y253H, E255K, M244V, M351T, F317V and H369P mutant lines were treated with ABPI alone, ASC alone, and in combination by serial dilution based starting from the measured IC50 concentration, and the Dose-Response matrix was calculated and analyzed through SynergyFinder. Results A part of the results are summarized in the figure. As shown, with each ABPI at the baseline IC50 concentration in K562 cells, cell growth was inhibited to 59.8%, 56.9%, 61.5%, 59.9% and 71.6-79.4% with IMA, BOS, DAS, NIL and ASC (Fig A). When cell growth inhibition was assessed with combination treatment of reduced dose of ABPI and fixed dose of ASC (3nM), we observed that the combination treatment of ASC with ABPI can inhibit BaF3 cell growth even at a reduced concentration of ABPI from IC50 (Fig B). When combined of ABPI at baseline doses with 3nM ASC, 100% of cell inhibition was noted as expected. When treated with 3nM ASC with with 50% reduced dose of ABPI, 84.6-100% of cell inhibition was noted in WT, 99.2-99.6% in G250E, 96.8-99.4% in E255K mutant BaF3 cell lines, concluding that these cell lines were highly sensitive to double blockade; M351T and F317V BaF3 cell lines showed in the range between 30-77% of cell inhibition with a half dose of ABPI/baseline dose ASC, referring it intermediate sensitive cell lines; H369P, Y253F and M244V cell lines showed less effective cell inhibition compared to other cell lines, indicating the low sensitive cell lines. It implies that each cell line does have its own inherent profile of resistance to double blockade (Fig C). In addition, DAS and BOS seems to be better ABPIs for double blockade for intermediate to low sensitive mutations except the F317V mutation, which can be overcome with IMA or NIL combination. Conclusion The current study strongly suggests that 1) double blockade with reduced dose of ABPI in combination with fixed dose of ASC is effective to control CML cells with different ABL1 KD mutations. Each cell lines does have its inherent resistance level to double blockades, which needs to be further explored. A phase 1 study with reduced dose of ABPI in combination with fixed dose ASC is strongly warranted to define an optimal dose for combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Finding a Needle in Haystack: Digital-PCR As a Sensitive Tool for MRD Detection in the Circulating-Tumor DNA Samples in Hodgkin Lymphoma

Introduction: Hodgkin lymphoma (HL) is the most frequent malignancy in young adults. The high probability of the cure is redeemed by both acute and long-term treatment sequelae. Balancing toxicity and efficacy requires highly reliable decision-making tools. Current treatment tailoring is based on the positron-emission tomography (PET/CT) assessment. The way how to improve the sensitivity of imaging methods could be a detection of minimal residual disease (MRD) in the circulating-tumor DNA (ctDNA) samples. The standard of the detection method is a next-generation sequencing (NGS), which covers a large spectrum of mutations, but is time-consuming, costly, and requires a significant concentration of ctDNA. The new digital PCR (dPCR) is a faster, cheaper method, which needs a lower amount of ctDNA. Aim: To validate the reliability of the dPCR for target-mutations of STAT6 and XPO1 genes detection in the peripheral blood ctDNA of patients with Hodgkin lymphoma Methods: CtDNA samples were obtained at the time of diagnosis/relapse, at the time of PET/CT assessment (interim, final), and during follow-up - every 3-6 months. CfDNA was extracted from peripheral blood plasma using the QiaAmp Circulating nucleic acid kit (Qiagen). Specific NGS panel covering coding sequences (including UTRs) of 13 selected genes (B2M, CD36, CIITA, GNA13, HIST1H1E, ITPKB, NFKBIE, PTPN1, SOCS1, SPEN, STAT6, TNFAIP3, and XPO1) was designed. For library preparation, we used SureSelect XT HS2 technology (Agilent Technologies) based on "target enrichment" with molecular barcodes. Sequencing was performed on a NovaSeq6000 (Illumina). Data were analyzed with the SureCall software (Agilent Technologies) with the sensitivity of 1.0% VAF. The detected variants were annotated using COSMIC, dbSNP, Ensembl, and ClinVar. Selected variants STAT6 p.N417Y and XPO1 p.E571K were further monitored by dPCR (QIAcuity Digital PCR System; Qiagen) with the sensitivity of 0.1% VAF. For dPCR experiments, we used a QIAcuityTM probe PCR kit (Qiagen) and nanoplate QIAcuity Nanoplate 26K 24-well (Qiagen). Primers and probes were taken and modified according to Camus et al., 2016 (EXPO1 p.E571K) and Spina et al., 2018 (STAT6 p.N417Y). All pts were analyzed with the same Siemens Biograph mCT scanner and results were independently reviewed by two physicians blinded for clinical outcome. Results: We have analyzed ctDNA using an NGS panel in 48 pts with CHL (44 at diagnosis, 4 in relapse). Mutations were detected in 22/48 (46%) pts. The most frequently mutated genes were STAT6 (12/48 pts), TNFAIP3 (10/48 pts), XPO1 (7/48 pts), and SOCS1 (7/26 pts). Frameshift deletions prevailed in TNFAIP3 and SOCS1 genes. Hotspot mutations in the STAT6 (p.N417Y, in five pts) and XPO1 (p.E571K, in six pts) genes we used as target mutations for dPCR cohort. The first date of ctDNA analysis was the first CHL diagnosis in ten and 1st relapse in one case. This dPCR-cohort (n=11) consists of 9 females and 2 males with a median age at the time of diagnosis of 40 yrs, with disease subtypes of nodular sclerosis (8), mixed-cellularity (1), lymphocyte-rich (1), and CHL unclassified (1). Mean VAF at diagnosis was 9.14% (0.76-25.0), mean cell-free DNA concentration 1.47 ng/mL (0.76-2.88). Three of the pts were considered to have intermediate and eight advanced cHL stages according to the GHSG system. Pts have received ABVD (n=5) or BEACOPPesc (n=5) or platinum-based+BEAM200 (n=1) protocols. Interim PET-2 was positive in 1 pt (stable disease), and final PET was positive in 2 (both PD). dPCR captured mutation in all three PETpos (0.12, 0.86, and 2.22%) cases, whereas was negative (<0.1% VAF) in all PETneg cases. During follow-up, two patients relapsed (both with detectable mutation, illustrative case is a Figure 1), in one case (pt during the BV post-ASCT maintenance) we observed transient dPCR positivity without evidence of relapse. Conclusion: A combination of the NGS (large-scale mutation screening) at the diagnosis with rapid and sensitive mutation monitoring using dPCR at the time of the treatment-tailoring or follow-up could improve decision-making accuracy in the routine care of Hodgkin lymphoma patients. Further analyses are needed to directly compare the sensitivity of the NGS and dPCR methods. Acknowledgement: Supported by MZCR-RVO (FNOL, 00098892) and AZV NU22-03-0018 grants. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract PR013: High sensitive mutation detection in liquid biopsy with duodenal fluid captures genetic mutations associated with pancreatic carcinogenesis

Abstract Background: Blood-based liquid biopsy for comprehensive genomic profiling is available for cancer patients; however, given the limited amount of tumor-derived DNA in plasma, it remained challenging to detect pancreatic ductal adenocarcinoma (PDA) at earlier stages. Here, we evaluated the mutation detection in DNA from the duodenal fluid as an alternative biospecimen obtained by a less-invasive approach relative to endoscopic biopsy. Methods: In 300 patients of PDA, IPMN, or other pancreatic diseases in seven institutions, we collected duodenal fluid containing pancreatic juice from the duodenal lumen through the endoscopic channel. Plasma was also collected, and DNA was extracted from both samples, and molecular barcode sequencing, which enabled the highly sensitive detection of low- frequent mutation, targeting five pancreatic cancer-associated genes, namely, KRAS, GNAS, TP53, CDKN2A, and SMAD4, were performed. Detected mutations were evaluated for concordance with surgically resected tissue or needle biopsy. Results: DNA extracted from the duodenal fluids (DFDNA) had a much higher yield (avg. 25.4 ng/ml) than plasma cell-free DNA (avg. 1.3 ng/ml). While plasma cfDNA had a typical fragmentation pattern with a peak at 160 bp, DFDNA has a variety of fragmentation patterns. Hotspot mutations detected in the duodenal fluids showed as much as 51.4% concordance with a 0.2 % the limit of detection (LOD) for its variant allele frequency in PDA-associated mutations in the DFDNA (n=109) with the mutations in PDA lesion, whereas only 25% concordance with 0.4% LOD in the plasma cfDNA (n=104). In contrast to plasma cfDNA, where liquid-tissue matches were limited to advanced stages of cancer, DFDNA assay enabled the higher concordance through a wide range of PDA stages, even Stage 0 (83.3%) or I (45.5%) UICC 8th, or IPMN cases, and allowed to detect of additional mutations that were originated from multicentric/concomitant lesions in the pancreas. Conclusions: Duodenal fluid-derived DNA can be a potent biospecimen to detect mutations with higher yield and sensitivity than plasma cfDNA. Although validation of the duodenal fluid collection and methods up to more sensitive analysis is required to meet the standards of practical testing, it has the potential to detect fast-stage pancreatic disease and the detection of lesions that were not visible on pathological images and is expected to lead to the implementation of highly sensitive liquid biopsy. Citation Format: Yusuke Ono, Kenji Takahashi, Hidenori Karasaki, Yusuke Mizukami. High sensitive mutation detection in liquid biopsy with duodenal fluid captures genetic mutations associated with pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR013.

A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance.

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

369P Comparison of the efficacy and safety of icotinib with vinorelbine or without vinorelbine as first-line treatment for advanced lung adenocarcinoma in patients with sensitive EGFR mutations: A retrospective study

EGFR-TKIs offer significantly greater efficacy than chemotherapy in patients with TKI sensitive EGFR mutations, TKIs are now the first line therapy for advanced lung adenocarcinoma in patients with sensitive EGFR mutations. Despite the high efficacy of these kinase inhibitors, radical cure of stage IV NSCLC remains an unachieved goal because invariably, almost all patients develop acquired resistance within one to several years. Constant efforts have been made to search strategy for overcoming resistance to kinase inhibitors, such as combined anti-vascular or combined chemotherapy.