Independent Resistance Mechanisms Research Articles

Insights into existing and futuristic treatment approach for chronic myeloid leukaemia

Oncogenes play a crucial part in human cancer development, and when particular drugs obstruct the proteins produced by these oncogenes, the tumoural process can be ceased. For instance, in chronic myeloid leukaemia (CML), all pathological traits are associated with a single oncogene, BCR-ABL1. CML is a triphasic cancerous disorder of haematopoietic stem cells, marked by a balanced translocation between chromosomes 9 and 22, leading to the genesis of a Philadelphia chromosome encompassing the BCR-ABL1 fusion gene. This fusion oncogene further produces a constitutive active tyrosine kinase protein, enhancing the downstream signalling pathways and constitutes cancer. The treatment for CML has been entirely altered from chemotherapy and immunotherapy to targeted therapy with the emergence of tyrosine kinase inhibitors (TKIs) which inhibit BCR-ABL1 kinase activity. However, the inhibitory mechanism of TKIs is constrained by BCR-ABL1 dependent and independent resistance mechanisms, prompting the exploration of novel therapeutics through extensive clinical trials to develop next-generation drugs with enhanced potency. The persistent challenges posed by CML have motivated researchers to seek innovative strategies for its eradication, such as the application of the genome editing tool CRISPR/Cas9. This review provides insights into existing CML diagnoses, treatment modalities, resistance mechanisms, drugs under trial phases and new potential therapeutic drugs. Furthermore, the review looks ahead to a visionary perspective wherein the CRISPR/Cas9 approach holds the potential to evolve into a prospective curative measure for CML.

Resistance mechanisms of EGFR tyrosine kinase inhibitors, in EGFR exon 20 insertion-mutant lung cancer

BackgroundMobocertinib, an EGFR exon 20 insertion (Ex20ins)–specific tyrosine kinase inhibitor has been used for treatment of advanced/metastatic EGFR Ex20ins-mutant non-small cell lung cancer (NSCLC). However, resistance mechanisms to EGFR Ex20ins-specific inhibitors and the efficacy of subsequent amivantamab treatment is unknown. MethodsTo investigate resistance mechanisms, tissue and cfDNA samples were collected before treatment initiation and upon development of resistance from NSCLC patients with EGFR Ex20ins mutations received mobocertinib, poziotinib, and amivantamab treatments. Genetic alterations were analyzed using whole-genome and targeted sequencing, and in vitro resistant cell lines were generated for validation. ResultsEGFR amplification (n = 6, including 2 broad copy number gain) and EGFR secondary mutation (n = 3) were observed at the resistance of mobocertinib. One patient had both EGFR secondary mutation and high EGFR focal amplification. In vitro models harboring EGFR alterations were constructed to validate resistance mechanisms and identify overcoming strategies to resistance. Acquired EGFR-dependent alterations were found to mediate resistance to mobocertinib in patients and in vitro models. Furthermore, two of six patients who received sequential amivantamab followed by an EGFR tyrosine kinase inhibitor had MET amplification and showed partial response. ConclusionsOur study revealed EGFR-dependent and -independent mechanisms of mobocertinib resistance in patients with advanced EGFR Ex20ins-mutant NSCLC.

Functional assessment of cadherin as a shared mechanism for cross/dual resistance to Cry1Ac and Cry2Ab in Helicoverpa zea

Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1, Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) in the United States. Laboratory-selected Cry1Ac/Cry2Ab cross resistance and field-evolved practical dual resistance of H. zea to these two toxins have been widely reported. Whether the widespread Cry1Ac/Cy2Ab dual resistance of H. zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear. Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species. To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab, we cloned H. zea cadherin (HzCadherin) cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain- and loss-of-function analyses. Heterologous expression of HzCadherin in H. zea midgut, H. zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab, whereas silencing HzCadherin of H. zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab. Likewise, suppressing HzCadherin with siRNA made H. zea larvae resistant to Cry1Ac. These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab, and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab.

Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence

BackgroundNovel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy.MethodsWe applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection.ResultsST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer-associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell marker expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to the tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic responses, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient.ConclusionsThese data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

The role of genetic testing in the prognosis and management of solid tumors. A literature review

Abstract Introduction: Cancer is the leading cause of death and an important impediment to increasing life expectancy in every country of the world. During the process of oncogenesis, genetic and epigenetic changes lead to abnormal expression of genes associated with cellular pathways that coordinate extremely important functions such as cell multiplication, cell differentiation, cell death, and cell cycle. Methods: There are over 200 approved biomarker-driven drugs for various types of cancer. Valuable biomarkers are analyzed to establish their importance in specific therapies. Precision medicine for oncological patients has been recognized as a valuable approach to solid tumors. Results: Various genes and their mutations either have a direct pathogenic effect or can give hints to a certain prognosis regarding the oncological pathology. A comprehensive genetic test for a broad molecular profile and complete characterization of tumor genetic heterogenicity should contain genes that are aligned with professional practice, guidelines and clinical trials, full coding region coverage for each gene and targeting of unique emerging and actionable markers. It is useful to use such a comprehensive test when a broad genomic profile identifies treatment options including immunotherapies and targeted drugs for patient enrollment or when relapse or disease progression has occurred after prior therapies. Conclusions: For patients with solid tumors, personalized medicine has been recognized as a successful strategy treatment, but it is not sufficient to seize cancer growth and progression up to a single molecular alteration due to specific hallmarks such as tumor heterogeneity, clonal evolution, and independent resistance mechanisms. Earlier studies have evaluated the effectiveness of using multigene panel screening methods for personalized cancer therapy, with controversial results. Future research in the field of circulating tumor DNA analysis might be the key to overcoming some of these limitations. The liquid biopsy could enable dynamic molecular profiling of all patients diagnosed with solid tumors enhancing accuracy, prognosis, and management

BCR-ABL Independent Resistance Mechanisms to Chronic Myeloid Leukemia

BCR-ABL Independent Resistance Mechanisms to Chronic Myeloid Leukemia

Genomic landscape and efficacy of HER2-targeted therapy in patients with HER2-mutant non-small cell lung cancer.

HER2-targeted therapy provides survival benefits to HER2-mutant non-small cell lung cancer (NSCLC). A better understanding of the clinical and genomic characterization of treatment-naïve HER2-positive NSCLC, as well as the efficacy of and resistance to HER2-targeted therapy in HER2-altered NSCLC, could promote further improvement of HER2 targeted therapy. HER2-altered NSCLC patients was retrospectively included and their genomic profiles were performed by next-generation sequencing. The clinical outcomes included overall response rate, disease control rate and progression-free survival. Among 176 treatment-naïve patients with HER2 alterations, 64.8% harbored HER2 mutations with/without HER2 amplification, and 35.2% carried HER2 amplification only. Molecular characterization was correlated with tumor stage that late-stage NSCLC with HER2 oncogenic mutations showed a higher prevalence of TP53 mutations and a higher tumor mutation burden. However, this correlation was not found in patients with HER2 amplification only. Twenty-one patients with HER2 alterations treated with pyrotinib or afatinib were retrospectively enrolled. Pyrotinib yielded a longer median progression-free survival than afatinib (5.9 [95% CI, 3.8-13.0] vs. 4.0 months [95% CI, 1.9-6.3], P = 0.06) in these patients. Analysis of the genomic profiles before and after anti-HER2 targeted therapies identified de novo HER2 copy number gain and G518W mutation, as well as mutations involving DNA damage repair signaling, SWI-SNF complex, and epigenetic regulations as potential resistance mechanisms. HER2-mutant NSCLC had different molecular features from HER2-amplified NSCLC, and its genomic profile was dependent of tumor stage. Pyrotinib had superior therapeutic effects than afatinib in HER2-altered NSCLC, although larger cohorts are warranted to validate it. HER2-dependent and -independent resistance mechanisms to afatinib and pyrotinib were unveiled.

Multiomic Analysis of CD38 Expression Levels in 209 Untreated and Daratumumab-Resistant Patients

The anti-CD38 antibody Daratumumab (Dara) is widely used in first-line as well as in relapsed/refractory multiple myeloma (MM). Primary or secondary resistance eventually occurs in all treated patients but the underlying mechanisms are poorly understood: Genetic alterations that lead to loss of function affecting epitopes on CD38, low baseline expression of CD38 or the selection of CD38dim cells were associated with suboptimal response to treatment. Interestingly, drugs such as all-trans retinoic acid, panobinostat or DNMT inhibitors increase Dara efficiency through reversible upregulation of CD38. Overall, three cohorts with overall 209 patients were examined for this dataset. We conducted flow cytometry in 21 therapy naïve and 19 Dara-refractory patients using a Dara epitope-specific antibody. All untreated and 11 of the resistant patients stained positive for CD38 MM, suggesting CD38 independent resistance mechanisms. In eight patients, CD38 expression was not detected; however, in five of them, additional staining with a CD38 multiepitope antibody confirmed CD38 persistence, reflecting epitope blockade through Dara or epitope mutation that interferes with the binding of the antibody. Finally, in three out of 19 refractory patients, CD38 could not be detected with both antibodies. To quantify CD38 receptor density on a molecular level, we established super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in the same patients. dSTORM uses fluorophore-conjugated antibodies and provides an unprecedented molecular 20 nm resolution for individual cells. We observed intra-patient heterogeneity of the CD38 receptor density with a mean expression of 35.15 ± 13.42 receptors/µm² (range 3.61-67.24) in the Dara naïve patients. Notably, in the refractory group, we found a significant decline (p=0.03) with a mean of 22.7 ± 18.26 receptors/µm² (range 17.07-63.77). In two of the three patients with no CD38 detectable, CD38 persistence could be assessed by dSTORM below the detection rate of flow cytometry. One patient remained negative for CD38, suggesting true loss of the receptor, causing Dara resistance in this patient. Next, we confirmed downregulation of CD38 gene expression using qPCR, and significantly reduced expression in the Dara refractory cohort (p=0.01). Of note, correlation between gene and receptor expression was moderate (p=0.49, r=0.57), leaving room for epigenetic regulation or posttranscriptional modification. To interrogate whether epigenetic regulation of CD38 via DNA methylation may underlie differential CD38 receptor expression, we performed deep bisulfite sequencing of MM cells and PBMCs obtained from 41 patients from our institution. CD38 on chromosome 4 contains several regulatory regions: One promoter, two adjacent promoter flanking regions and 11 enhancer regions described in ENSEMBL genome browser (GRCh38.13). The CD38 promoter was fully unmethylated (mean methylation: 0.31% ± 0.2%, range 0.2%-0.9%), but variable methylation of a CD38 promoter flanking region upstream of the promoter was detectable with 30.08% ± 17.8% (range 0.9%-61.5%) in the MM cells compared to 59.6% ± 3.9% (range 49.0%-66.7%) in the PBMCs from the same patients (p <0.001). This highlights phenotypic plasticity and suggests that DNA methylation may play a regulatory role in CD38 expression. We are currently investigating the methylome of all 14 regulatory regions in 38 Dara resistant patients (18 of them with longitudinal sampling before and after Dara therapy), 33 Dara responsive patients and 52 plasma cells obtained from healthy donors. Finally, in a cohort of 127 patients from our institution with WGS data available we screened for genetic alterations with potential impact on the CD38 receptor expression. We found monoallelic CD38 deletions in 18 patients, with one patient having an additional 11kb deletion conferring to an exon 3 loss. Exon-6 skipping, as recently described, was not detectable in the cohort. In conclusion, genetic alterations affecting the CD38 locus and true loss of the CD38 receptor are rare events and do not explain Dara resistance in most affected patients. dSTORM imaging provides additional insights in the CD38 receptor downregulation in MM patients treated with anti-CD38 moAb therapy. Regulatory mechanisms such as epigenetic regulation need to be further explored.

Therapeutic Targeting of FLT3 in Acute Myeloid Leukemia: Current Status and Novel Approaches.

FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients. The presence of FLT3-ITD (internal tandem duplication, 20-25%) mutation and, to a lesser extent, FLT3-TKD (tyrosine kinase domain, 5-10%) mutation is associated with poorer diagnosis and therapy response since the leukemic cells become hyperproliferative and resistant to apoptosis after continuous activation of FLT3 signaling. Targeting FLT3 has been the focus of many pre-clinical and clinical studies. Hence, many small-molecule FLT3 inhibitors (FLT3is) have been developed, some of which are approved such as midostaurin and gilteritinib to be used in different clinical settings, either in combination with chemotherapy or alone. However, many questions regarding the best treatment strategy remain to be answered. On the other hand, various FLT3-dependent and -independent resistance mechanisms could be evolved during FLT3i therapy which limit their clinical impact. Therefore, identifying molecular mechanisms of resistance and developing novel strategies to overcome this obstacle is a current interest in the field. In this review, recent studies of approved FLT3i and knowledge about major resistance mechanisms of clinically approved FLT3i's will be discussed together with novel treatment approaches such as designing novel FLT3i and dual FLT3i and combination strategies including approved FLT3i plus small-molecule agents targeting altered molecules in the resistant cells to abrogate resistance. Moreover, how to choose an appropriate FLT3i for the patients will be summarized based on what is currently known from available clinical data. In addition, strategies beyond FLT3i's including immunotherapeutics, small-molecule FLT3 degraders, and flavonoids will be summarized to highlight potential alternatives in FLT3-mutated AML therapy.

Novel Dicarboximide BK124.1 Breaks Multidrug Resistance and Shows Anticancer Efficacy in Chronic Myeloid Leukemia Preclinical Models and Patients' CD34+/CD38- Leukemia Stem Cells.

Simple SummaryChemotherapy is a first line treatment in many cancer types, but the constant exposition to chemotherapeutics often leads to therapy resistance. An example is chronic myeloid leukemia that, due to the use of tyrosine kinase inhibitors such as imatinib, remains manageable, however incurable. Overall, 20–25% of imatinib responders develop secondary resistance, and among them, 20–40% is due to mechanisms such as expression of P-glycoprotein (MDR1) or leukemia stem cells’ mechanisms of survival and cancer regrowth. This study provides the first evidence from animal and cellular models that this resistance can be overcome with the novel dicarboximide BK124.1. The compound causes no visible toxicity in mice, and has proper pharmacokinetics for therapeutic applications. It was efficient against both multidrug resistant CML blasts and CD34+/CD38− leukemia stem cells coming from CML patients. Future development of BK124.1 could offer curative treatment of CML and of other cancers resistant or intolerant to current chemotherapy.The search is ongoing for new anticancer therapeutics that would overcome resistance to chemotherapy. This includes chronic myeloid leukemia, particularly suitable for the studies of novel anticancer compounds due to its homogenous and well-known genetic background. Here we show anticancer efficacy of novel dicarboximide denoted BK124.1 (C31H37ClN2O4) in a mouse CML xenograft model and in vitro in two types of chemoresistant CML cells: MDR1 blasts and in CD34+ patients’ stem cells (N = 8) using immunoblotting and flow cytometry. Intraperitoneal administration of BK124.1 showed anti-CML efficacy in the xenograft mouse model (N = 6) comparable to the commonly used imatinib and hydroxyurea. In K562 blasts, BK124.1 decreased the protein levels of BCR-ABL1 kinase and its downstream effectors, resulting in G2/M cell cycle arrest and apoptosis associated with FOXO3a/p21waf1/cip1 upregulation in the nucleus. Additionally, BK124.1 evoked massive apoptosis in multidrug resistant K562-MDR1 cells (IC50 = 2.16 μM), in CD34+ cells from CML patients (IC50 = 1.5 µM), and in the CD34+/CD38− subpopulation consisting of rare, drug-resistant cancer initiating stem cells. Given the advantages of BK124.1 as a potential chemotherapeutic and its unique ability to overcome BCR-ABL1 dependent and independent multidrug resistance mechanisms, future development of BK124.1 could offer a cure for CML and other cancers resistant to present drugs.

Utility of Next-Generation Sequencing in the Reconstruction of Clonal Architecture in a Patient with an EGFR Mutated Advanced Non-Small Cell Lung Cancer: A Case Report

EGFR tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, targeted therapies impose a strong selective pressure against the coexisting tumor populations that lead to the emergence of resistant clones. Molecular characterization of the disease is essential for the clinical management of the patient, both at diagnosis and after progression. Next-generation sequencing (NGS) has been established as a technique capable of providing clinically useful molecular profiling of the disease in tissue samples and in non-invasive liquid biopsy samples (LB). Here, we describe a case report of a patient with metastatic NSCLC harboring EGFR mutation who developed two independent resistance mechanisms (EGFR-T790M and TP53 + RB1 mutations) to dacomitinib. Osimertinib given as a second-line treatment eliminated the EGFR-T790M population and simultaneously consolidated the proliferation of the TP53 + RB1 clone that eventually led to the histologic transformation to small-cell lung cancer (SCLC). Comprehensive NGS profiling revealed the presence of the TP53 + RB1 clone in the pretreatment biopsy, while EGFR-T790M was only detected after progression on dacomitinib. Implementation of NGS studies in routine molecular diagnosis of tissue and LB samples provides a more comprehensive view of the clonal architecture of the disease in order to guide therapeutic decision-making.

Treatment patterns after poly-ADP ribose polymerase (PARP) inhibitors in epithelial ovarian cancer patients

ObjectivesThe primary objective of this study was to describe treatment patterns after poly-ADP ribose polymerase (PARP) inhibitor in patients with epithelial ovarian cancer. Secondary objectives were to evaluate duration of...

Experimental Determination of Cancer Drug Targets with Independent Mechanisms of Resistance.

Mathematical modelling of tumour mutation dynamics has suggested that cancer drug targets that have different resistance mechanisms should be good candidates for combination treatment. This is because the development of mutations that cause resistance to all drugs at once should arise relatively infrequently. However, it is difficult to identify drug targets fulfilling this requirement for particular cancers. Here we present four experimental criteria that we argue are necessary (but not sufficient) conditions that drug combinations should meet in order to be considered for combination drug treatment aimed at delaying or overcoming cancer drug resistance. We present the results of our own experiments - guided by these criteria - using anaplastic lymphoma kinase mutated lung cancer cells. Each set of experiments demonstrate results for different drug combinations. We conclude that the combination of ALK and MEK inhibitors come closest to meeting all our criteria.

Combatting acquired resistance to osimertinib in EGFR-mutant lung cancer.

The discovery of activating mutations in epidermal growth factor receptor (EGFR) in non-small-cell lung cancer transformed the care and prognosis of patients and heralded the era of 'personalized medicine' in thoracic oncology. Osimertinib, a third-generation EGFR inhibitor, has been established as the preferred EGFR inhibitor for newly diagnosed patients which urged the need to develop treatment options for patients progressing on first-line osimertinib. However, acquired resistance invariably emerges and numerous efforts have been attempted to delay or overcome acquired resistance. In this article, we thoroughly reviewed the current understanding of osimertinib resistance mechanisms and explored the established and emerging treatment options. Newer treatment strategies targeting EGFR-dependent or -independent resistance mechanisms, novel approaches using bispecific antibodies and antibody-drug conjugates will be discussed. Moreover, what to do with brain only progression, and how to incorporate immunotherapy in EGFR-mutant lung cancer will be discussed. Lastly, future perspectives on the ongoing clinical trials and combination of front-line therapy will be introduced.

Quantitative Insights Into β-Lactamase Inhibitor's Contribution in the Treatment of Carbapenemase-Producing Organisms With β-Lactams.

Objectives: Carbapenemase-producing organisms (CPOs) are associated with high mortality rates. The recent development of β-lactamase inhibitors (BLIs) has made it possible to control CPO infections safely and effectively with β-lactams (BLs). This study aims to explicate the quantitative relationship between BLI’s β-lactamase inhibition and CPO’s BL susceptibility restoration, thereby providing the infectious disease society practical scientific grounds for regulating the use of BL/BLI in CPO infection treatment.Methods: A diverse collection of human CPO infection isolates was challenged by three structurally representative BLIs available in the clinic. The resultant β-lactamase inhibition, BL susceptibility restoration, and their correlation were followed quantitatively for each isolate by coupling FIBA (fluorescence identification of β-lactamase activity) and BL antibiotic susceptibility testing.Results: The β-lactamase inhibition and BL susceptibility restoration are positively correlated among CPOs under the treatment of BLIs. Both of them are dependent on the target CPO’s carbapenemase molecular identity. Of note, without sufficient β-lactamase inhibition, CPO’s BL susceptibility restoration is universally low across all tested carbapenemase molecular groups. However, a high degree of β-lactamase inhibition would not necessarily lead to a substantial BL susceptibility restoration in CPO probably due to the existence of non-β-lactamase BL resistance mechanisms.Conclusion: BL/BLI choice and dosing should be guided by quantitative tools that can evaluate the inhibition across the entire β-lactamase background of the CPO upon the BLI administion. Furthermore, rapid molecular diagnostics for BL/BLI resistances, especially those sensitive to β-lactamase independent BL resistance mechanisms, should be exploited to prevent ineffective BL/BLI treatment.

Abstract 37: Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples

Abstract Determining patterns and mechanisms of drug resistance is fundamentally required for improving clinical outcome of cancer treatments. The ability to study multiple samples from different metastatic sites of the same patient is a clinically challenging task, which has become possible with the advent of “rapid” autopsy procedures (&amp;lt;6 hours from death) conducted on the patients deceased from cancer. We have obtained and analyzed whole-exome, whole genome and transcriptome sequencing data from advanced breast cancer samples derived from multiple cancer lesions of the same patient post-treatment through the Massachusetts General Hospital Rapid Autopsy program. We have integrated genomic and transcriptomic data through advanced clonal reconstruction methods (PhylogicNDT) to investigate how the patients developed resistance to multiple lines of anti-cancer therapy. Additionally, we have used previously collected cell-free DNA samples to establish both a spatial and temporal picture of cancer drug resistance and progression. We have examined clonal heterogeneity and resistance to targeted therapies in 10 metastatic ER+ breast cancer patients over 120 distinct metastatic sites. The majority of patients in this cohort were treated with CDK4/6 cell cycle inhibitors in addition to hormone therapy. We discovered that multiple metastatic sites often share similar clonal structure, but overall progeny of several sibling clones spreads throughout the body, creating “families” of more closely related lesions. These distinct lesion populations often develop independent resistance mechanisms to the many lines of treatment that the patents have received. At least 8 out of 10 patients showed multiple known and potentially novel resistance mechanisms in separate branches of the phylogenetic tree, often converging on distinct mutations in the same resistance genes. Notably we have identified ESR1, KRAS and chromatin modifier recurrent and convergent mutations across the cohort. Temporal analysis using cfDNA taken over the course of treatment allows us to identify clones that expanded in response to a given treatment. Using RNA expression and pathway analysis, we identify unique transcriptional programs, differentially expressed genes and ER-signaling changes between distinct clones within one patient, as well as compare genetically similar clones across patents. Citation Format: Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Elizaveta Leshchiner, Thomas Zhang, Christopher Pinto, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Laxmi Parida, Gad Getz, Dejan Juric. Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 37.

Usefulness of BioFire FilmArray BCID2 for Blood Culture Processing in Clinical Practice.

ABSTRACTRapid pathogen characterization from positive blood cultures (BC) can improve management of patients with bloodstream infections (BSI). The FilmArray blood culture identification (BCID) assay is a molecular test approved for direct identification of BSI causing pathogens from positive BC. A recently updated version of the panel (BCID2) comprises improved species identification characteristics and allows for the detection of one expanded-spectrum β-lactamase (ESBL)- and several carbapenemase-encoding genes. Here, the clinical performance of the BCID2 assay for species identification in 180 positive BCs was evaluated. BCID2 results were concordant with the standard of care (SOC) in 159/180 (88.3%) BCs; 68/74 (91.9%) and 71/74 (96.0%) of all samples growing monobacterial, Gram-positive or Gram-negative pathogens, respectively, were identified, in agreement with SOC results. Nonconcordance was related to the detection of additional pathogens by the BCID2 assay (n = 4), discrepant species identification (n = 4), or failure of BCID2 to detect on-panel pathogens (n = 1). A number (12/31; 38.7%) of discordant results became evident in polymicrobial BC specimens. BCID2 identified the presence of blaCTX-M-carrying species in 12 BC specimens but failed to predict third-generation cephalosporin resistance in four isolates exhibiting independent cephalosporin resistance mechanisms. Carbapenem resistance related to the presence of blaVIM-2 or blaOxa-48-like was correctly predicted in two isolates. In conclusion, the BCID2 assay is a reliable tool for rapid BC processing and species identification. Despite inclusion of common ESBL- or carbapenemase-encoding markers, the multifactorial nature of β-lactam resistance in Gram-negative organisms warrants combination of BCID2 with (rapid) phenotypic susceptibility assays.

Mechanisms and markers of resistance to androgen signaling inhibitors in patients with metastatic castration-resistant prostate cancer

Next-generation androgen signaling inhibitors such as abiraterone and enzalutamide are widely used for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, baseline and acquired resistance to these treatments is commonly observed. In the last few years, significant effort has been devoted to uncover the molecular mechanisms and predictive markers of resistance. These analyses identified various DNA (single nucleotide variations, amplifications) and RNA variants (e.g., the splice variant AR-V7) of androgen receptor in association with resistance to abiraterone and enzalutamide therapies. Additionally, androgen receptor independent resistance mechanisms were also described. Some of these alterations can be detected in tumor tissues and/or in liquid biopsies of prostate cancer patients and therefore may serve as predictive biomarkers. According to the diversity of potential resistance mechanisms, it appears that a combination of markers representing various resistance mechanisms may provide better performance as single markers. In the present review, we summarize the most important androgen receptor dependent and independent resistance mechanisms and pay attention to methodological details. Recent data has highlighted that some of the resistance mechanisms to next-generation antiandrogen agents are associated with a better response to other therapies, we give an overview on currently ongoing clinical studies evaluating this promising aspect.

A first approach to develop a quantitative screening method to identify resistance to bacterial leaf spot disease caused by Acidovorax anthurii in anthurium

Resistance of anthurium cultivars to infection by Acidovorax anthurii, the causal agent of bacterial leaf spot disease was investigated using seven inoculation methods, four inoculum concentrations and two different plant ages towards developing a screening method for identification of disease resistance. Injection of 1 mL of 1.5 × 109 CFU mL−1 three-quarter way down the intact petiole from the intact leaf lamina of a stage-4 leaf consistently and repeatedly elicited 100% infection in susceptible anthurium cultivars, and was the best inoculation method. All 9 measures of resistance using the optimized inoculation method were able to differentiate among 14 anthurium cultivars. Of the three most discriminating measures, proportion of infected leaves per plant showed the largest correlations with field resistance measures obtained through a farm survey and a field trial conducted in a sick plot, and was the best measure of resistance. The study provided evidence for two independent mechanisms of resistance, one at the level of entry and infection establishment and the other at the level of severity of infection. Only two anthurium cultivars showed resistance at both levels. In addition to a cultivar dependent mechanism of resistance, the study showed that there is a general mechanism of mature plant resistance. The study provides a quantitative screening method that can be used to breed for resistance to A. anthurii in anthurium.

A Burkholderia thailandensis DedA Family Membrane Protein Is Required for Proton Motive Force Dependent Lipid A Modification.

The DedA family is a conserved membrane protein family found in most organisms. A Burkholderia thailandensis DedA family protein, named DbcA, is required for high-level colistin (polymyxin E) resistance, but the mechanism awaits elucidation. Modification of lipopolysaccharide lipid A with the cationic sugar aminoarabinose (Ara4N) is required for colistin resistance and is dependent upon protonmotive force (PMF) dependent transporters. B. thailandensis ΔdbcA lipid A contains only small amounts of Ara4N, likely leading to colistin sensitivity. Two B. thailandensis operons are required for lipid A modification with Ara4N, one needed for biosynthesis of undecaprenyl-P-Ara4N and one for transport of the lipid linked sugar and subsequent lipid A modification. Here, we directed overexpression of each arn operon by genomic insertion of inducible promoters. We found that overexpression of arn operons in ΔdbcA can partially, but not completely, restore Ara4N modification of lipid A and colistin resistance. Artificially increasing the PMF by lowering the pH of the growth media also increased membrane potential, amounts of Ara4N, and colistin resistance of ΔdbcA. In addition, the products of arn operons are essential for acid tolerance, suggesting a physiological function of Ara4N modification. Finally, we show that ΔdbcA is sensitive to bacitracin and expression of a B. thailandensis UppP/BacA homolog (BTH_I1512) can partially restore resistance to bacitracin. Expression of a different UppP/BacA homolog (BTH_I2750) can partially restore colistin resistance, without changing the lipid A profile. This work suggests that maintaining optimal membrane potential at slightly alkaline pH media by DbcA is responsible for proper modification of lipid A by Ara4N and provides evidence of lipid A modification-dependent and -independent mechanisms of colistin resistance in B. thailandensis.