Multiple Resistance Mutations Research Articles

Abstract LB389: Highly potent and selective RET inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations

Abstract RET gene mutations and fusions are the driving events for the generation and deterioration of many cancer types. The first generation RET inhibitors, selpercatinib (Loxo-292) and pralsetinib (Blu-667), demonstrated clinically acquired resistance (e.g., solvent frontier mutations G810X, double mutations V804X-G810X etc.). Novel treatment that overcomes multiple resistance mutations is needed to improve patient outcomes. Leveraging our in-house AI technology platform, we have discovered the next generation RET inhibitors that overcome multiple resistance mutations including solvent front, gatekeeper and various double mutations. We employ the synergistic advantages of domain expertise and modern technology to accelerate early stage drug discovery. On our deep learning (DL) centric AI platform, extensive scientific computing supports large scale virtual screening, generative design, drug property prediction and multi-objective lead optimization. Only the optimized candidate compounds are passed along for synthesis and testing to save time and cost. In cell proliferation assays, our compound effectively inhibited KIF5B-RET Ba/F3 WT, V804M, G810S, G810R, V804M-G810S with IC50 values of 0.4 nM, 1.3 nM, 1.1 nM, 3.4 nM, 6.3 nM, respectively, demonstrating significant improvement over selpercatinib in the range of several hundred fold in activities. Importantly, the compound is highly selective against VEGFR2, an off-target responsible for cardiovascular adverse effects in the approved RET inhibitors. The superior cellular potencies translated consistently into animal models. In Ba/F3 KIF5B-RET-G810R xenograft model, treatment of 25 mg/kg bid resulted in >90% TGI with a100% survival rate. The compound showed favorable pharmacokinetic properties, lack of drug-drug interaction (DDI), and devoid of hERG liability (IC50 > 10uM). Preclinical safety evaluations in rats exhibited good tolerance margin. We have discovered the second generation compound that potently inhibits a diverse range of RET alterations. The compound exhibits preferable pharmaceutical profiles and is advancing toward IND application. Citation Format: Li Xing, Wei Zhu, Jingbing Wang. Highly potent and selective RET inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB389.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019–2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Ultra-deep mutational landscape in chronic lymphocytic leukemia uncovers dynamics of resistance to targeted therapies.

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

Multidrug-resistant mutations to antiviral and antibody therapy in an immunocompromised patient infected with SARS-CoV-2

Antiviral and antibody therapies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being recommended for high-risk patients, but the potential for the development of multidrug-resistant mutations in immunocompromised patients is unclear. To investigate the treatment course in cases of prolonged viral shedding in an immunocompromised patient with SARS-CoV-2 infection, we conducted longitudinal measurements of laboratory tests, chest computed tomography (CT) image evaluations, antibody titers, and antigen levels in nasopharyngeal swabs. Furthermore, we performed whole-genome sequencing and digital PCR analysis to examine the mechanisms of drug resistance. We present a case of a 65-year-old man with a history of malignant lymphoma who was treated with multiple antiviral and antibody therapies, including sotrovimab, remdesivir, paxlovid (nirmatrelvir/ritonavir), and molnupiravir. Initially, viral antigen levels decreased after treatments. However, after the virus rebounded, the patient showed no virologic response. The viral genome analysis revealed a single Omicron subvariant (BA.1.1), which evolved within the host during the disease progression. The viruses had acquired multiple resistance mutations to nirmatrelvir (3 chymotrypsin-like protease [3CLpro] E166 A/V), sotrovimab (spike P337L and E340K), and remdesivir (RNA-dependent RNA polymerase [RdRp] V166L). Our results indicate that viruses with multidrug-resistant mutations and survival fitness persist in the infected subpopulation after drug selection pressure. This study was supported by the JSPS KAKENHI Early-Career Scientists 18K16292 (Y.H.), Grant-in-Aid for Scientific Research (B) 20H03668 and 23H02955 (Y.H.), the YASUDA Medical Foundation (Y.H.), the Uehara Memorial Foundation (Y.H.), the Takeda Science Foundation (Y.H.), and Kato Memorial Bioscience Foundation (Y.H.).

The host plant strongly modulates acaricide resistance levels to mitochondrial complex II inhibitors in a multi-resistant field population of Tetranychus urticae

The two-spotted spider mite Tetranychus urticae is a polyphagous pest with an extraordinary ability to develop acaricide resistance. Here, we characterize the resistance mechanisms in a T. urticae population (VR-BE) collected from a Belgian tomato greenhouse, where the grower was unsuccessful in chemically controlling the mite population resulting in crop loss. Upon arrival in the laboratory, the VR-BE population was established both on bean and tomato plants as hosts. Toxicity bioassays on both populations confirmed that the population was highly multi-resistant, recording resistance to 12 out of 13 compounds tested from various mode of action groups. DNA sequencing revealed the presence of multiple target-site resistance mutations, but these could not explain resistance to all compounds. In addition, striking differences in toxicity for six acaricides were observed between the populations on bean and tomato. The highest difference was recorded for the complex II inhibitors cyenopyrafen and cyflumetofen, which were 4.4 and 3.3-fold less toxic for VR-BE mites on tomato versus bean. PBO synergism bioassays suggested increased P450 based detoxification contribute to the host-dependent toxicity. Given the involvement of increased detoxification, we subsequently determined genome-wide gene expression levels of VR-BE on both hosts, in comparison to a reference susceptible population, revealing overexpression of a large set of detoxification genes in VR-BE on both hosts compared to the reference. In addition, a number of mainly detoxification genes with higher expression in VR-BE on tomato compared to bean was identified, including several cytochrome P450s. Together, our work suggests that multi-resistant field populations can accumulate a striking number of target-site resistance mutations. We also show that the host plant can have a profound effect on the P450-associated resistance levels to cyenopyrafen and cyflumetofen.

Bacteria exposed to antiviral drugs develop antibiotic cross-resistance and unique resistance profiles

Antiviral drugs are used globally as treatment and prophylaxis for long-term and acute viral infections. Even though antivirals also have been shown to have off-target effects on bacterial growth, the potential contributions of antivirals to antimicrobial resistance remains unknown. Herein we explored the ability of different classes of antiviral drugs to induce antimicrobial resistance. Our results establish the previously unrecognized capacity of antivirals to broadly alter the phenotypic antimicrobial resistance profiles of both gram-negative and gram-positive bacteria Escherichia coli and Bacillus cereus. Bacteria exposed to antivirals including zidovudine, dolutegravir and raltegravir developed cross-resistance to commonly used antibiotics including trimethoprim, tetracycline, clarithromycin, erythromycin, and amoxicillin. Whole genome sequencing of antiviral-resistant E. coli isolates revealed numerous unique single base pair mutations, as well as multi-base pair insertions and deletions, in genes with known and suspected roles in antimicrobial resistance including those coding for multidrug efflux pumps, carbohydrate transport, and cellular metabolism. The observed phenotypic changes coupled with genotypic results indicate that bacteria exposed to antiviral drugs with antibacterial properties in vitro can develop multiple resistance mutations that confer cross-resistance to antibiotics. Our findings underscore the potential contribution of wide scale usage of antiviral drugs to the development and spread of antimicrobial resistance in humans and the environment.

Abstract 5590: Liquid biopsy testing in metastatic or advanced breast cancer patients during the COVID-19 pandemic

Abstract Objective: During the COVID-19 pandemic, cancer patients had restricted access to standard of care tissue biopsy. Liquid biopsy assays using next generation sequencing technology provides a less invasive method for determining circulating tumour mutations (ctDNA) associated with targeted treatments or prognosis. As part of deploying technology to help cancer patients obtain molecular testing, a clinical program was initiated to offer liquid biopsy testing for Canadian patients with advanced or metastatic breast cancer. Methods: Blood was drawn in two 10 mL StreckTM DNA BCTs and sent to the CAP/CLIA/DAP accredited Imagia Canexia Health laboratory for testing using the clinically validated Follow ItTM liquid biopsy assay. Plasma was isolated using a double spin protocol and plasma cell-free DNA (cfDNA) extracted using an optimized Promega Maxwell RSC method. Extracted cfDNA was amplified using the multiplex amplicon-based hotspot 30 or 38 gene panel and sequenced. An in-house developed bioinformatics pipeline and reporting platform were used to identify pathogenic single nucleotide variants (SNVs), indels (insertions and deletions), and gene amplification. Included in the panel are genes associated with metastatic breast cancer: AKT1, BRAF, ERBB2, ESR1, KRAS, PIK3CA, TP53. Results: To identify biomarkers, 1214 metastatic or advanced breast cancer patient cfDNA samples were tested. There were 15 cases sent for repeat testing. We reported 48% of samples harboring pathogenic ctDNA mutations in TP53 (22%), PIK3CA (19%), ESR1 (18%), AKT1 (2%), ERBB2 (1.5%). Co-occurring variants were identified in samples with ESR1/PIK3CA as well as TP53/PIK3CA (both p-values <0.001). Interestingly, 29% of samples with mutated ESR1 harbored ≥ 2 ESR1 ctDNA mutations. In 56% of cases, previous molecular testing indicated the cancer subtype as hormone receptor (ER, PR) positive with/without HER2 negative status. In this specific subgroup, 49% harbored ctDNA mutations with 63% of those being PIK3CA and/or ESR1 mutations. Conclusions: A population of Canadian women with metastatic breast cancer were tested using a liquid biopsy gene panel during the COVID-19 pandemic for identification of biomarkers for targeted therapeutic options. Over 50% of the samples were identified as hormone positive, with greater than 60% harboring PIK3CA and ESR1 ctDNA mutations. Studies have shown that metastatic PIK3CA mutated ER-positive/HER2-negative tumors are predictive to respond to alpelisib therapy and have FDA and Health Canada approval. Additionally, ESR1 mutations are associated with acquired resistance to antiestrogen therapies, and interestingly we identified 29% of ESR1 mutated samples with multiple mutations possibly indicating resistance subclones. In future studies, longitudinal monitoring for presence of multiple targetable and resistance mutations could be utilized to predict or improve clinical management. Citation Format: Benjamin L. Furman, Ebru Baran, Sonal Brahmbhatt, Betty Chan, Ka Mun Nip, Adrian Kense, Brenda Murphy, Vincent Funari, David G. Huntsman, Ruth Miller, Melissa K. McConechy. Liquid biopsy testing in metastatic or advanced breast cancer patients during the COVID-19 pandemic. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5590.

Abstract 99: Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance

Abstract Tumors frequently acquire resistance to therapy resulting in disease relapse and patient mortality. Therapeutic approaches to overcome acquired resistance are hindered by limited treatment options and the presence of multiple parallel resistance mechanisms. Rather than treating resistance after it emerges, it may possible to prevent it by inhibiting the adaptive processes which initiate resistance. However, these processes and the genes which control them are poorly understood. Here we report that drug-tolerant cancer persister cells, which constitute residual disease and seed relapse, undergo drug stress-induced sublethal apoptotic signaling resulting in activation of apoptotic DNase DFFB without cell death. DFFB induces DNA damage and mutagenesis in persister cells, revealing DFFB as a key regulator of stress-induced mutagenesis in persister cells. Furthermore, we found in multiple tumor types that DFFB is required for acquired resistance to targeted therapies. Mechanistically, DFFB induces multiple putative resistance mutations and may also promote epigenetic changes which result in resistance. These results reveal DFFB as a promising potential therapeutic target to prevent acquired resistance. In addition, this stress-sensing mutagenic mechanism may promote mutagenesis in other contexts including normal tissues because sublethal apoptotic signaling may result from a variety of physiological stresses. Citation Format: August Finley Williams, David Gervasio, Claire Turkal, Anna Stuhlfire, Michael Wang, Brandon Mauch, Ariel West, Michelle Paw, Mehrshad Hairani, Cooper Lathrop, Sophie Harris, Jennifer Page, Matthew Hangauer. Apoptotic DNase DFFB mediates cancer persister cell mutagenesis and acquired drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 99.

Babesia microti Variant With Multiple Resistance Mutations Detected in an Immunocompromised Patient Receiving Atovaquone Prophylaxis.

We report Babesia microti genomic sequences with multiple mutations in the atovaquone-target region of cytochrome b, including a newly identified Y272S mutation, plus 1 mutation of undetermined significance in the azithromycin-associated ribosomal protein L4. The parasite was sequenced from an immunocompromised patient on prophylactic atovaquone for Pneumocystis pneumonia before diagnosis of babesiosis.

A high-throughput KASP assay provides insights into the evolution of multiple resistant mutations in populations of the two-spotted spider mite Tetranychus urticae across China.

The two-spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target-site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021. The KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2-6 and 7-10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy-Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection. Our study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple-acaricides resistance. © 2023 Society of Chemical Industry.

392P A phase II study of SAF-189s in patients with advanced ROS1 fusion-positive non-small cell lung cancer

ROS1 fusions (ROS1+) are enriched in 1-2% of non-small cell lung cancer (NSCLC) cases. SAF-189s is a novel, next-generation ALK/ROS1 inhibitor which overcomes multiple resistance mutations. We explored the efficacy and safety of SAF-189s in phase (Ph) 2 study in patients (pts) with ROS1 fusion NSCLC, with or without ROS1 inhibitor treatment.

47 (PB037) - TYRA-200: Potent Against FGFR2 Fusions, Molecular Brake Mutations and Gatekeeper Resistance

Background: Approved pan-FGFR (fibroblast growth factor receptor) inhibitors and those in late-stage clinical development (pemigatinib, infigratinib, and futibatinib) have demonstrated a clinical benefit in metastatic FGFR2-fusion or rearranged intrahepatic cholangiocarcinoma (ICC). However, inhibition of emerging polyclonal on-target acquired resistance mutations remains a critical unmet need. TYRA-200 is an FGFR1/2/3 inhibitor that was designed to specifically address these clinically observed acquired resistance mutations within the kinase domain of FGFR2. A significant therapeutic benefit may be achieved from this precision approach for FGFR2-driven cancers. Materials and Methods: TYRA-200 was evaluated in enzymatic assays and cell lines driven by FGFR2 fusions and mutations. in vivo tumor growth inhibition with TYRA-200 was tested in several FGFR2-driven models. Results: In enzymatic assays, TYRA-200 maintained potent inhibition for the gatekeeper mutants V565L/F and the molecular brake mutants N550D/H/ K and E566A. Cellular potency for these mutants was confirmed in a panel of Ba/F3 cell lines expressing wild type FGFR2 and variants N550 K, V565F, V565I, K660E and K660N. Additionally, TYRA-200 maintained potency in vitro in an endometrial cancer cell line AN3CA, which harbors the molecular brake mutation N550 K. in vivo tumor regressions were achieved in both the AN3CA and Ba/F3 FGFR2 V565F models. In the AN3CA xenograft model 80% tumor regression was achieved, while in the Ba/F3 FGFR2 V565F allograft model, a 64% tumor regression was seen, whereas tumor regression was not observed with futibatinib. Conclusions: TYRA-200 is currently under development for FGFR2-altered advanced solid tumors, including ICC. Importantly, the data demonstrates that TYRA-200 retains potency across multiple resistance mutations which emerge during therapy, including gatekeeper and molecular brake mutations. Conflict of interest: Ownership: Tyra Biosciences shareholders.

Chinese Expert Consensus on Management of Special Adverse Effects Associated with Lorlatinib

间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）融合基因是非小细胞肺癌（non-small cell lung cancer, NSCLC）中第二常见的肿瘤驱动基因。作为新型的第三代ALK酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI），洛拉替尼对多种ALK激酶域突变具有广谱且高效的临床活性，并具有强大的穿透血脑屏障效力。洛拉替尼的总体耐受性良好，其独特的不良反应或不良事件包括高脂血症与中枢神经系统反应等，多为轻至中度，通常经剂量调整和/或标准医疗干预即可管理。对于ALK阳性晚期NSCLC，开始洛拉替尼治疗前应充分评估患者基线特征与既往用药状况，预先告知患者可能经历的用药相关不良反应，并定期监测以实现药物临床获益的最大化。同时，多学科专家团队对于建立全面的诊断和治疗策略是至关重要的。

Lineage dynamics in growing biofilms: Spatial patterns of standing vs. de novo diversity.

Microbial biofilms show high phenotypic and genetic diversity, yet the mechanisms underlying diversity generation and maintenance remain unclear. Here, we investigate how spatial patterns of growth activity within a biofilm lead to spatial patterns of genetic diversity. Using individual-based computer simulations, we show that the active layer of growing cells at the biofilm interface controls the distribution of lineages within the biofilm, and therefore the patterns of standing and de novo diversity. Comparing biofilms of equal size, those with a thick active layer retain more standing diversity, while de novo diversity is more evenly distributed within the biofilm. In contrast, equal-sized biofilms with a thin active layer retain less standing diversity, and their de novo diversity is concentrated at the top of the biofilm, and in fewer lineages. In the context of antimicrobial resistance, biofilms with a thin active layer may be more prone to generate lineages with multiple resistance mutations, and to seed new resistant biofilms via sloughing of resistant cells from the upper layers. Our study reveals fundamental “baseline” mechanisms underlying the patterning of diversity within biofilms.

Conformational adjustment overcomes multiple drug-resistance mutants of tropomyosin receptor kinase

Mutation-induced resistance to targeted drug treatment poses a serious threat to successful chemotherapy. Multiple mutations underlying drug resistance remain a largely unsolved scientific issue. Tropomyosin receptor kinases (TRKs) are promising therapeutic targets for several malignant human cancers, but they have become less effective due to multiple resistance mutations. Thus, TRKs are representative cases to explore the problem of multiple resistance mutations. Here, we proposed a conformational adjustment strategy of drug design to overcome multiple resistance mutations in cancer treatments. A representative inhibitor, TIY-7, exhibited remarkable inhibitory activity against five TRK mutants, showing an IC50 value of 1.1 nM against the most severe mutant TRKA-G595R. Moreover, it displayed superior tumor growth inhibitory activity compared with the clinically used drug selitrectinib. These results validated our strategy to design a new inhibitor structure to overcome multiple resistance mutations.

Genomic insights into the evolution of Echinochloa species as weed and orphan crop

As one of the great survivors of the plant kingdom, barnyard grasses (Echinochloa spp.) are the most noxious and common weeds in paddy ecosystems. Meanwhile, at least two Echinochloa species have been domesticated and cultivated as millets. In order to better understand the genomic forces driving the evolution of Echinochloa species toward weed and crop characteristics, we assemble genomes of three Echinochloa species (allohexaploid E. crus-galli and E. colona, and allotetraploid E. oryzicola) and re-sequence 737 accessions of barnyard grasses and millets from 16 rice-producing countries. Phylogenomic and comparative genomic analyses reveal the complex and reticulate evolution in the speciation of Echinochloa polyploids and provide evidence of constrained disease-related gene copy numbers in Echinochloa. A population-level investigation uncovers deep population differentiation for local adaptation, multiple target-site herbicide resistance mutations of barnyard grasses, and limited domestication of barnyard millets. Our results provide genomic insights into the dual roles of Echinochloa species as weeds and crops as well as essential resources for studying plant polyploidization, adaptation, precision weed control and millet improvements.

Real-world circulating tumor DNA analysis depicts resistance mechanism and clonal evolution in ALK inhibitor-treated lung adenocarcinoma patients

BackgroundSequential treatment with different generations of anaplastic lymphoma kinase (ALK) inhibitors have been widely applied to ALK-positive lung cancer; however, resistance mutations inevitably developed. Further characterization of ALK resistance mutations may provide key guidance to subsequent therapies. Here we explored the emergence of secondary ALK mutations during sequential ALK tyrosine kinase inhibitor (TKI) treatment in a real-world study of Chinese lung adenocarcinoma (ADC) patients.MethodsA clinical-genomic database was queried for lung ADC patients with at least one ALK inhibitor treatment and at least one plasma sample collected following ALK inhibitor treatment. Targeted genome profiling was performed with a 139-gene panel in baseline tumor tissue and serial plasma samples of patients.ResultsA total of 116 patients met inclusion criteria. ALK G1202R was more common in patients with echinoderm microtubule-associated protein-like 4 (EML4)-ALK v3 fusion, whereas ALK L1196M was more common in v1. TP53 mutant patients were significantly associated with harboring multiple ALK resistance mutations (P = 0.03) and v3+/TP53 mutant patients had the highest rate of multiple ALK resistance mutations. The sequential use of ALK TKI led to an increased incidence of concurrent ALK mutations along the lines of therapies. Alectinib had a lower rate (9%) harboring ALK resistance mutation as first-line ALK TKI compared with crizotinib (36%). ALK compound mutations identified included ALK D1203N/L1196M, ALK G1202R/L1196M, and ALK G1202R/F1174C, which may be lorlatinib resistant. Using paired pretreatment and post-treatment samples, we identified several ALK-independent resistance-related genetic alterations, including PTPRD and CNKN2A/B loss, MYC, MYCN and KRAS amplification, and EGFR19del.ConclusionsSequential postprogression plasma profiling revealed that increased lines of ALK inhibitors can accelerate the accumulation of ALK resistance mutations and may lead to treatment-refractory compound ALK mutations. The selection for optimal first-line TKI is very important to achieve a more efficacious long-term strategy and prevent the emergence of on-target resistance, which may provide guidance for clinical decision making.

Target-site resistance to trifluralin is more prevalent in annual ryegrass populations from Western Australia.

Trifluralin is widely used in Australia as one of the important pre-emergence herbicides to control annual ryegrass (Lolium rigidum Gaud.) populations. Trifluralin resistance evolution and mechanisms have been identified in some ryegrass populations. In this study, 21 putative resistant field survey populations from Western Australian were screened with trifluralin, and 90% (19 of 21) contained individuals surviving 480 g ha-1 trifluralin treatment. Twelve populations contained individuals possessing the known α-tubulin resistance mutations at Val-202, Thr-239 and Arg-243 in TUA4 (alpha-tubulin 4 n), plus multiple potential resistance mutations in TUA4 pending genetic confirmation. Three populations had only individuals carrying newly identified (but uncharacterized) mutations in TUA3/TUA4. Radioactive work found that six populations evolved metabolic resistance to trifluralin, and at least four of them also possessed the known and/or putative target-site mutations. These results confirm that a high incidence of resistance to the dinitroaniline herbicide (trifluralin) is present, and target-site tubulin mutations make a major contribution to resistance in these annual ryegrass populations. Co-evolution of both target-site and non-target-site resistance to per-emergence herbicides warrants diverse management tactics.

Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors

AbstractThe genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.

Monitoring Circulating Tumor DNA During Surgical Treatment in Patients with Gastrointestinal Stromal Tumors.

The majority of patients diagnosed with advanced gastrointestinal stromal tumors (GISTs) are successfully treated with a combination of surgery and tyrosine kinase inhibitors (TKIs). However, it remains challenging to monitor treatment efficacy and identify relapse early. Here, we utilized a sequencing strategy based on molecular barcodes and developed a GIST-specific panel to monitor tumor-specific and TKI resistance mutations in cell-free DNA and applied the approach to patients undergoing surgical treatment. Thirty-two patients with GISTs were included, and 161 blood plasma samples were collected and analyzed at routine visits before and after surgery and at the beginning, during, and after surgery. Patients were included regardless of their risk category. Our GIST-specific sequencing approach allowed detection of tumor-specific mutations and TKI resistance mutations with mutant allele frequency < 0.1%. Circulating tumor DNA (ctDNA) was detected in at least one timepoint in nine of 32 patients, ranging from 0.04% to 93% in mutant allele frequency. High-risk patients were more often ctDNA positive than other risk groups (P < 0.05). Patients with detectable ctDNA also displayed higher tumor cell proliferation rates (P < 0.01) and larger tumor sizes (P < 0.01). All patients who were ctDNA positive during surgery became negative after surgery. Finally, in two patients who progressed on TKI treatment, we detected multiple resistance mutations. Our data show that ctDNA may become a clinically useful biomarker in monitoring treatment efficacy in patients with high-risk GISTs and can assist in treatment decision making.