Early Development Of Resistance Research Articles

TP53 gain-of-function mutations promote osimertinib resistance via TNF-α–NF-κB signaling in EGFR-mutated lung cancer

EGFR tyrosine kinase inhibitors (TKIs) are effective against EGFR-mutated lung cancer, but tumors eventually develop resistance to these drugs. Although TP53 gain-of-function (GOF) mutations promote carcinogenesis, their effect on EGFR-TKI efficacy has remained unclear. We here established EGFR-mutated lung cancer cell lines that express wild-type (WT) or various mutant p53 proteins with CRISPR-Cas9 technology and found that TP53-GOF mutations promote early development of resistance to the EGFR-TKI osimertinib associated with sustained activation of ERK and expression of c-Myc. Gene expression analysis revealed that osimertinib activates TNF-α–NF-κB signaling specifically in TP53-GOF mutant cells. In such cells, osimertinib promoted interaction of p53 with the NF-κB subunit p65, translocation of the resulting complex to the nucleus and its binding to the TNF promoter, and TNF-α production. Concurrent treatment of TP53-GOF mutant cells with the TNF-α inhibitor infliximab suppressed acquisition of osimertinib resistance as well as restored osimertinib sensitivity in resistant cells in association with attenuation of ERK activation and c-Myc expression. Our findings indicate that induction of TNF-α expression by osimertinib in TP53-GOF mutant cells contributes to the early development of osimertinib resistance, and that TNF-α inhibition may therefore be an effective strategy to overcome such resistance in EGFR-mutant lung cancer with TP53-GOF mutations.

Mutational Dynamics Related to Antibiotic Resistance in M. tuberculosis Isolates from Serial Samples of Patients with Tuberculosis and Type 2 Diabetes Mellitus.

Genetic variation in tuberculosis is influenced by the host environment, patients with comorbidity, and tuberculosis-type 2 diabetes mellitus (TB-T2DM) and implies a higher risk of treatment failure and development of drug resistance. Considering the above, this study aimed to evaluate the influence of T2DM on the dynamic of polymorphisms related to antibiotic resistance in TB. Fifty individuals with TB-T2DM and TB were initially characterized, and serial isolates of 29 of these individuals were recovered on day 0 (diagnosis), 30, and 60. Genomes were sequenced, variants related to phylogeny and drug resistance analyzed, and mutation rates calculated and compared between groups. Lineage X was predominant. At day 0 (collection), almost all isolates from the TB group were sensitive, apart from four isolates from the TB-T2DM group showing the mutation katG S315T, from which one isolate had the mutations rpoB S450L, gyrA A90G, and gyrA D94G. This pattern was observed in a second isolate at day 30. The results provide a first overview of the dynamics of mutations in resistance genes from individuals with TB-T2DM, describing an early development of resistance to isoniazid and a rapid evolution of resistance to other drugs. Although preliminary, these results help to explain the increased risk of drug resistance in individuals with TB and T2DM.

Abstract C143: Unveiling synergistic interplay between KRASG12C inhibitors and the polyamine synthesis inhibitor SAM486 against lung cancer cells: An in vitro study

Abstract Introduction: Non-small cell lung cancer (NSCLC) represents the most lethal variant of lung cancer. The G12C mutation in the KRAS protein occurs in 20-30% of NSCLC patients and is correlated with an unfavorable prognosis. Adagrasib and sotorasib are emerging compounds that inhibit the KRASG12C protein, demonstrating effectiveness in NSCLC driven by KRASG12C. However, the early development of resistance to KRASG12C inhibitors emphasizes the necessity to investigate combined therapeutic approaches. Polyamines (putrescine, spermidine, and spermine) are essential molecules for cancer growth and survival and are elevated in various cancer types, including NSCLC. Adenosylmethionine decarboxylase-1 (AMD1) is a critical enzyme involved in spermidine and spermine synthesis. AMD1 depends on KRAS signaling via the IGF-AKT-mTOR signaling and can be selectively restrained by the compound SAM486. Aim: This study aimed to explore the effect of AMD1 inhibition in NSCLC cells using the compound SAM486, alone or in combination with the KRASG12C inhibitors adagrasib and sotorasib, and to investigate the relationship between KRASG12C inhibition and polyamine enzyme levels. Methods: We employed four NSCLC cell lines: H358 (KRASG12C), A549 (KRASG12S), CORL-L23 (KRASG12V), and H1299 (KRASwt). Immunoblotting analysis was utilized to measure AMD1 levels and KRAS-ERK pathway phosphorylation. Cell proliferation was assessed through BrdU incorporation and colony formation assays. Cell viability was determined by MTT reduction, and combination studies were evaluated using Combenefit and SynergyFinder software, employing the Loewe additivity model. Results: The AMD1 inhibitor SAM486 exhibited a reduction in cell proliferation in NSCLC cells, irrespective of the presence of KRAS mutations. Furthermore, the KRASG12C inhibitors sotorasib and adagrasib decreased AMD1 levels and cell viability in H358 cells harboring the KRASG12C mutation. Notably, a high degree of synergy was observed when combining sotorasib and adagrasib with SAM486 in KRASG12C-mutated cells, as evaluated by Combenefit and SynergyFinder software, employing the Loewe additivity model. Conclusion: These findings suggest that the simultaneous inhibition of KRASG12C and AMD1 could provide a novel and synergistic therapeutic strategy against tumors with KRASG12C mutations. Citation Format: Rodrigo A Lopez-Muñoz, Antonia Martin-Martin, Carina Chipon, Neudo Buelvas, Sergio Guzmán-Kunstmann, Matías Mansilla-Velásquez. Unveiling synergistic interplay between KRASG12C inhibitors and the polyamine synthesis inhibitor SAM486 against lung cancer cells: An in vitro study [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C143.

Induction Regimen in High-Risk Neuroblastoma: A Pilot Study of Highly Effective Continuous Exposure of Tumor Cells to Radio-Chemotherapy Sequence for 1 Month. The Critical Role of Iodine-131-Metaiodobenzylguanidine.

Simple SummaryDespite the use of intensive chemotherapy, the prognosis of high-risk neuroblastoma continues to be dismal. Higher cure rates appear to be mainly correlated with the response to induction therapy. Nevertheless, in recent decades, there has been no significant improvement in the response rate to most induction treatments. The present induction regimen, of only one month duration, which includes iodine-131-metaiodobenzylguanidine (i.e., low-dose rate form of irradiation that persists in the tumor cells for a few weeks) allows for a highly effective continuous exposure of tumor cells to both chemotherapy and radiotherapy, at the time of maximal tumor cell sensitivity to treatment. Following future investigations, along the same line, a higher cure rate in patients with advanced neuroblastoma might be achieved.The prognosis of high-risk neuroblastoma (NB) continues to be poor. The early development of resistance often leads to disease recurrence. In the present study, an innovative induction regimen, including an intensive initial radio-chemotherapy sequence based on the use of iodine-131-metaiodobenzylguanidine (131-I-MIBG), was investigated. The duration of the regimen lasted only one month. Fifteen newly diagnosed patients aged >18 months with high-risk NB were treated with cisplatin, etoposide, cyclophosphamide, and vincristine, followed on day 10 by 131-I-MIBG (dose: 12–18.3 mCi/kg). Cisplatin and vincristine were administered on day 20 and 21 followed by the re-administration of vincristine, cyclophosphamide, and doxorubicin on day 29 and 30. Non-hematologic toxicity was not observed. Moderate hematologic toxicity was present probably attributable to chemotherapy. The evaluation of response was performed approximately 50 days after the initiation of treatment, yielding four complete responses, eight very good partial responses, one partial response, and two non-responses. Importantly, a complete metastatic response was achieved in 87% of patients. The present pilot study, which includes 131-I-MIBG, allows for a highly effective continuous exposure of tumor cells to both chemotherapy and radiotherapy. Furthermore, early high-dose chemotherapy followed by stem cell rescue may achieve high levels of tumor cell clearance and improve the prognosis of high-risk NB.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma: A Physiopathologic and Pharmacologic Review.

Simple SummaryPDAC is one of the most malignant tumors and its treatment, whether surgery or chemotherapy, has shown poor results. Resistance to gemcitabine and other chemotherapeutic drugs is an essential factor in this failure. This review analyzes the molecular causes of gemcitabine resistance and discusses the possibilities of new approaches aimed at decreasing, delaying or even reversing chemoresistance in pancreatic cancer.Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with a poor prognosis and inadequate response to treatment. Many factors contribute to this therapeutic failure: lack of symptoms until the tumor reaches an advanced stage, leading to late diagnosis; early lymphatic and hematic spread; advanced age of patients; important development of a pro-tumoral and hyperfibrotic stroma; high genetic and metabolic heterogeneity; poor vascular supply; a highly acidic matrix; extreme hypoxia; and early development of resistance to the available therapeutic options. In most cases, the disease is silent for a long time, andwhen it does become symptomatic, it is too late for ablative surgery; this is one of the major reasons explaining the short survival associated with the disease. Even when surgery is possible, relapsesare frequent, andthe causes of this devastating picture are the low efficacy ofand early resistance to all known chemotherapeutic treatments. Thus, it is imperative to analyze the roots of this resistance in order to improve the benefits of therapy. PDAC chemoresistance is the final product of different, but to some extent, interconnected factors. Surgery, being the most adequate treatment for pancreatic cancer and the only one that in a few selected cases can achieve longer survival, is only possible in less than 20% of patients. Thus, the treatment burden relies on chemotherapy in mostcases. While the FOLFIRINOX scheme has a slightly longer overall survival, it also produces many more adverse eventsso that gemcitabine is still considered the first choice for treatment, especially in combination with other compounds/agents. This review discusses the multiple causes of gemcitabine resistance in PDAC.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa

BackgroundNew insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.MethodsMosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin.ResultsLower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin (11.4‒51.9% mortality in Nkolondom, 31.7‒48.2% in Mangoum, 34.6‒56.1% in Mayuge, 39.4‒45.6% in Obuasi, 83.7‒89.3% in Congo and 71.1‒95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Similar observations were done for imidacloprid and acetamiprid. Synergist assays (PBO, DEM and DEF) with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR = 0.5; P = 0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR = 2.10; P = 0.013).ConclusionsThis study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone + MERO (4 µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.Graphical

Effectiveness of combination with eribulin in third line of chemotherapy for triple negative breast cancer (case report)

In most cases triple negative breast cancer is characterized by an aggressive course of disease and early development of resistance to chemotherapy. Thereafter, the late-line treatment choice, usually after anthracyclines and taxanes, is problematic due to the limited amount of effective and low-toxic cytostatics. In our opinion, in this situation the use of eribulin which possesses unique antitumor action mechanisms is a good option. An illustrative case of a pronounced antitumor effect of eribulin in metastatic breast cancer with triple negative phenotype resistant to previous lines of chemotherapy is presented.

Evidence to Date: Evaluating Pembrolizumab in the Treatment of Extensive-Stage Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive subtype of lung cancer characterized by a rapid initial response and early development of resistance to systemic therapy and radiation. The management of SCLC significantly changed for the first time in decades with the introduction of immune checkpoint inhibitors. Pembrolizumab, a humanized IgG4 isotype antibody, targets the programmed cell death protein 1 (PD-1) pathway to restore anti-tumor immunity. Prospective trials of pembrolizumab in patients with previously treated SCLC showed significant durability of responses. These results led to the U.S. Food and Drug Administration (FDA) granting pembrolizumab accelerated approval as second- or third-line monotherapy for patients with extensive-stage (ES) SCLC. In a recent clinical trial that included patients with previously untreated ES-SCLC, pembrolizumab in combination with platinum/etoposide met its progression-free survival endpoint, but overall survival (OS) did not cross the threshold for superiority. With the therapeutic landscape for SCLC rapidly evolving, we review prior experience and future directions of pembrolizumab in ES-SCLC.

Emerging antibody therapies for pancreatic adenocarcinoma: a review of recent phase 2 trials

ABSTRACT Introduction: Pancreatic adenocarcinoma is now the third-leading cause of cancer-related deaths in the US which can be attributed to rising incidence, diagnosis at advanced stages and early development of metastasis. Systemic therapy remains palliative with early development of resistance possibly related to the constitutive activation of ‘undruggable’ KRAS, immunosuppressive microenvironment, and intense desmoplasia. The advancements in molecular biology has led to the development and investigation of targeted and immune therapeutics. Areas covered: This study provides a comprehensive review of the literature to further the understanding of molecular targets with their respective antibody-based therapies in clinical development in pancreatic cancer. PubMed was systematically searched for English-language articles discussing antibody-based therapies under phase 2 clinical trial investigation in pancreatic adenocarcinoma. Expert opinion: PDAC remains highly resistant to chemotherapy with no significant improvement in survival for patients with advanced or metastatic cancer. Unfortunately, the majority of the antibody-based targeted and immune therapeutics have failed to meet their primary efficacy endpoints in early phase trials. However, there are a few promising antibody-based drugs with intriguing preliminary data that merit further investigation, while many more continue to be developed and investigated preclinically, and in early phase trials.

Detection and characterization of resistance to acetolactate synthase inhibiting herbicides in Anisantha and Bromus species in the United Kingdom.

Anisantha and Bromus spp. are widespread and difficult to control, potentially due to the evolution of herbicide resistance. In this study, UK populations of four brome species have been tested for the early development of resistance to acetolactate synthase (ALS)-inhibiting herbicides commonly used in their control. Glasshouse assays confirmed reduced sensitivity to ALS-inhibiting herbicides in single populations of A. diandra, B. commutatus and B. secalinus, and in three populations of A. sterilis. By contrast, all 60 brome populations tested were sensitive to the ACCase-inhibiting herbicide propaquizafop and glyphosate. Dose-response with two ALS herbicides showed broad-ranging resistance in the A. diandra, A. sterilis and B. commutatus populations. In the B. commutatus population, this was associated with a point mutation in the ALS enzyme conferring target site resistance (TSR). Additionally, resistant populations of A. sterilis and B. commutatus populations contained enhanced amounts of an orthologue of the glutathione transferase phi (F) class 1 (GSTF1) protein, a functional biomarker of nontarget site resistance (NTSR) in Alopecurus myosuroides. There was further evidence of NTSR as these plants also demonstrated an enhanced capacity to detoxify herbicides. This study confirms the evolution of resistance to ALS inhibiting herbicides in brome species in the UK by mechanisms consistent with the evolution of both TSR and NTSR. These findings point to the need for increased vigilance in detecting and mitigating against the evolution of herbicide resistance in brome species in Northern Europe. © 2020 Society of Chemical Industry.

Abstract C29: Development of a circulating tumor DNA assay in pancreas cancer

Abstract Pancreas cancer is the third most common cause of cancer-related death, owing in part to high rates of unresectable disease at time of diagnosis and poor long-term response to chemotherapy. Biomarkers are needed to sensitively identify and monitor disease burden, and to measure response to therapy. The CA19-9 tumor marker lacks sufficient positive or negative predictive value to guide treatment decisions, and digital droplet PCR (ddPCR) assessment of circulating KRAS mutations suffers poor sensitivity on account of only assessing a single DNA locus. We hypothesize that a multitargeted circulating tumor DNA assay can outperform CA19-9 and ddPCR as biomarker for the detection and monitoring of pancreas cancer. Our lab has developed the cancer personalized profiling by deep sequencing (CAPP-Seq) method, which combines high-depth sequencing with several strategies of error-suppression to identify minute amounts of tumor DNA circulating in patients’ blood. The development of our CAPP-Seq targeted sequencing panel hinged primarily on capturing regions of the genome known to be recurrently mutated in pancreatic ductal adenocarcinoma (PDAC), based on The Cancer Genome Atlas project. These regions contain mutations in KRAS, TP53, SMAD4, and by a long-tail of mutations in many other genes for which we contributed approximately 200 kb of selector space. In addition to recurrent SNVs, we also contributed approximately 35 kb for recurrent copy number variant locations, 10 kb for recurrent noncoding variants, and 20 kb for regions with aberrant expression patterns in PDAC based on TCGA RNA-sequencing data. This purpose of this study is to employ CAPP-Seq in two pancreas cancer cohorts: patients who underwent resection at Stanford and had blood drawn before and after surgical resection (n=30), and a cohort of patients with metastatic disease who are undergoing chemotherapy with blood draws at multiple time-points (n=20). The former cohort will assess prediction of recurrence based on ctDNA level, and the latter will assess patients with early development of resistance to chemotherapy. Before assessing this ctDNA detection assay in patients with pancreas cancer, we first evaluated the CAPP-Seq approach on two healthy research subjects and achieved on average 4000x de-duplicated sequencing depth, for a theoretical limit of detection of 0.03% circulating tumor fraction. We next sequenced samples from 7 patients with resectable disease and 8 with metastatic disease, with results expected in early July. Citation Format: Dan King, Mari Olsen, George Fisher, Max Diehn, Ash Alizadeh. Development of a circulating tumor DNA assay in pancreas cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C29.

Abstract B023: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors

Abstract Metastatic colorectal cancer (mCRC) harboring a BRAF(V600E) mutation is associated with poor prognosis and limited treatment options. Clinical trials targeting BRAF with MEK ± EGFR in 2- and 3-drug combinatorial therapy regimens have produced modest response rates and progression-free survival, with early development of resistance in most patients. Considerable efforts to study the mutational landscape or gene expression profiles or synthetic lethal genetic interactions of BRAF(V600E) mCRC have remained relatively ineffective at pinpointing novel therapeutic susceptibilities. This suggests that intrinsic resistance of BRAF(V600E) mCRC to combinatorial treatments is not driven by individual genetic dependencies, but is rather mediated by the concerted upregulation of multiple, parallel signaling pathways that cooperate to circumvent therapeutic effectiveness. To characterize which signaling circuits are rewired by BRAF and EGFR-targeted therapy, we used our new high throughput kinase activity mapping (HT-KAM) platform (Coppé et al 2019 Nature Cell Biology), which can reveal druggable kinase dependencies in cancer cell lines and tumor biospecimens by directly measuring the phospho-catalytic activity of kinases using their biological peptide targets as phospho-sensors. Out of the concerted, reprogrammed kinase circuits we identified with HT-KAM, we found a highly conserved, cell-autonomous, SRC kinases-relayed, COX2-PGE2-GNAS-driven inflammatory program that functions independently of the commonly studied BRAF-MEK-ERK / EGFR / PDPK1-AKT1 pathways. Specifically, we found that SRC family kinases are catalytically activated upon single and combination treatment with BRAF ± MEK ± EGFR inhibitors. We validated the specificity and potency of this BRAF/EGFR-independent vulnerability using genetic interventions (via shRNA or CRISPR) and drug treatments (adding dasatinib or saracatinib) in cell survival and colony formation assays across >15 BRAF(V600E) cell lines, and in patient tumor-derived xenograft (PDX) mouse models. Immunohistochemistry performed on patient tumors resistant to targeted therapies, and on residual PDX tumors after BRAF and MEK treatments, further reiterated SRC activation. Mechanistically, the activation of SRC kinases was induced by an autocrine PGE2-regulated GNAS-activation loop that COX2-inhibitors celecoxib or valdecoxib reversed both in vitro and in vivo. In PDX models, addition of celecoxib significantly improved tumor growth inhibition, and systematically outperformed 2- and 3-drug targeted therapy regimens tested in clinical trials without increasing toxicity (manuscript in preparation). In conclusion, we demonstrate that SRC-signaling is at the nexus of an autonomous inflammatory program with pro-tumorigenic activities, which may explain why BRAF(V600E) colorectal tumors develop resistance to current therapies. Moreover, targeting COX2 presents a promising new clinical strategy to restore therapeutic sensitivity in patients. Citation Format: Chloe E Atreya, Ana Ruiz-Saenz, Changjun Wang, Bo Pan, Courtney A Dreyer, Diede Brunen, Anirudh Prahallad, Danislav Spassov, Dana J Steffen, Byron C Hann, Scott R VandenBerg, Silvio Gutkind, Mark M Moasser, Laura J van 't Veer, Jean-Philippe Coppe. A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B023. doi:10.1158/1535-7163.TARG-19-B023

New β‑lactam antibiotics and β‑lactamase inhibitors against multidrug-resistant Gram-negative bacteria

The worldwide spread of multidrug-resistant Gram-negative bacteria (MDR-GN) continues. Treatment options for infections caused by MDR-GN remain scarce and only few new substances are currently in clinical phaseII/III studies or have already been granted market approval. To provide an overview about current data on new β‑lactam antibiotics and β‑lactamase inhibitor combinations, respectively. New macrolides, ketolides and aminoglycosides are not addressed. Selective literature research regarding published data on ceftazidim/avibactam, ceftolozan/tazobactam, imipenem/cilastatin + relebactam, meropenem/vaborbactam, aztreonam/avibactam and cefiderocol, as well as registered trials. The development of new antimicrobials for the treatment of MDR-GN infections offers new options for attending physicians. β‑Lactamase producers are inhibited by these new substances, though with varying efficacy; however, there are still no adequate treatment options for metallo-β-lactamase (MBL) producers. Clinical data are still indifferent and come from heterogeneous patient collectives. Direct comparisons with established treatment strategies, such as the "last-resort use" of polymyxins are hardly possible. Cases of early development of resistance have already been described. Finally, the importance of toxicity and optimal dosing-in organ failure or organ replacement procedures such as dialysis-remain unclear.

Vemurafenib plus cobimetinib in unresectable stage IIIc or stage IV melanoma: response monitoring and resistance prediction with positron emission tomography and tumor characteristics (REPOSIT): study protocol of a phase II, open-label, multicenter study

BackgroundIn patients with BRAFV600 mutated unresectable stage IIIc or metastatic melanoma, molecular targeted therapy with combined BRAF/MEK-inhibitor vemurafenib plus cobimetinib has shown a significantly improved progression-free survival and overall survival compared to treatment with vemurafenib alone. Nevertheless, the majority of BRAFV600 mutation-positive melanoma patients will eventually develop resistance to treatment.Molecular imaging with 18F-Fluorodeoxyglucose (18F-FDG) PET has been used to monitor response to vemurafenib in some BRAFV600 mutated metastatic melanoma patients, showing a rapid decline of 18F-FDG uptake within 2 weeks following treatment. Furthermore, preliminary results suggest that metabolic alterations might predict the development of resistance to treatment. 18F-Fluoro-3′-deoxy-3’L-fluorothymidine (18F-FLT), a PET-tracer visualizing proliferation, might be more suitable to predict response or resistance to therapy than 18F-FDG.MethodsThis phase II, open-label, multicenter study evaluates whether metabolic response to treatment with vemurafenib plus cobimetinib in the first 7 weeks as assessed by 18F-FDG/18F-FLT PET can predict progression-free survival and whether early changes in 18F-FDG/18F-FLT can be used for early detection of treatment response compared to standard response assessment with RECISTv1.1 ceCT at 7 weeks.Ninety patients with BRAFV600E/K mutated unresectable stage IIIc/IV melanoma will be included. Prior to and during treatment all patients will undergo 18F-FDG PET/CT and in 25 patients additional 18F-FLT PET/CT is performed. Histopathological tumor characterization is assessed in a subset of 40 patients to unravel mechanisms of resistance. Furthermore, in all patients, blood samples are taken for pharmacokinetic analysis of vemurafenib/cobimetinib. Outcomes are correlated with PET/CT-imaging and therapy response.DiscussionThe results of this study will help in linking PET measured metabolic alterations induced by targeted therapy of BRAFV600 mutated melanoma to molecular changes within the tumor. We will be able to correlate both 18F-FDG and 18F-FLT PET to outcome and decide on the best modality to predict long-term remissions to combined BRAF/MEK-inhibitors. Results coming from this study may help in identifying responders from non-responders early after the initiation of therapy and reveal early development of resistance to vemurafenib/cobimetinib. Furthermore, we believe that the results can be fundamental for further optimizing individual patient treatment.Trial registrationClinicaltrials.gov identifier: NCT02414750. Registered 10 April 2015, retrospectively registered.

Hepatitis C virus RNA levels at week-2 of telaprevir/boceprevir administration are predictive of virological outcome

BackgroundTriple therapy with telaprevir/boceprevir+pegylated-interferon+ribavirin can achieve excellent antiviral efficacy, but it can be burdened with resistance development at failure. AimsTo evaluate kinetics of hepatitis C virus (HCV) RNA decay and early resistance development, in order to promptly identify patients at highest risk of failure to first generation protease inhibitors. MethodsHCV-RNA was prospectively quantified in 158 patients receiving pegylated-interferon+ribavirin+telaprevir (N=114) or+boceprevir (N=44), at early time-points and during per protocol follow-up. Drug resistance was contextually evaluated by population sequencing. ResultsHCV-RNA at week-2 was significantly higher in patients experiencing virological failure to triple-therapy than in patients with sustained viral response (2.3 [1.9–2.8] versus 1.2 [0.3–1.7]logIU/mL, p<0.001). A 100IU/mL cut-off value for week-2 HCV-RNA had the highest sensitivity (86%) in predicting virological success. Indeed, 23/23 (100%) patients with undetectable HCV-RNA reached success, versus 26/34 (76.5%) patients with HCV-RNA<100IU/mL, and only 11/31 (35.5%) with HCV-RNA>100IU/mL (p<0.001). Furthermore, differently from failing patients, none of the patient with undetectable HCV-RNA at week-2 had baseline/early resistance. ConclusionsWith triple therapy based on first generation protease inhibitors, suboptimal HCV-RNA decay at week-2 combined with early detection of resistance can help identifying patients with higher risk of virological failure, thus requiring a closer monitoring during therapy.

Urinary tract infection in small outpatient children: the influence of age and gender on resistance to oral antimicrobials

Urinary tract infection (UTI) is a common bacterial disease in small children in which treatment with antimicrobials is used. The worldwide increase of bacterial resistance to these drugs is threatening the efficacy of such treatment and may increase the risk for long-term damage. The aim of this retrospective study was to analyse the development of resistance to oral antimicrobials over a 10-year period in an unselected outpatient population of small children with first-time UTI. The patient material included 494 boys and 512 girls below 2years of age with community acquired symptomatic UTI. Escherichia coli bacteria were isolated in 96% of girls and 89% of boys (p < 0.0001). The overall resistance of E. coli was 14% to trimethoprim and below 1% to cefadroxil and nitrofurantoin. Over the 10-year period, the trimethoprim resistance of E. coli increased from 5 to 17% but remained unchanged to cefadroxil and nitrofurantoin. E. coli resistance to trimethoprim was related to age: 11% below and 19% above 9months (p < 0.01). The increase in resistance over time and with age was found only in girls. The increasing resistance of E. coli to trimethoprim makes this drug less suitable for empiric treatment of UTI. Young children with UTI seem predisposed to early development of resistance. Therefore, surveillance of resistance to antimicrobials with special regard to age and gender is recommended.

Abstract C41: BRAFV600 serum/plasma analysis: Predictive value of survival in melanoma treated with BRAF inhibitors.

Abstract Background: BRAF mutation is present in 50% of metastatic melanoma patients. Treatment with BRAF inhibitors improves survival of BRAF mutant melanoma patients, with a median progression free survival time of 6 months. Twenty percent of patients are refractory to BRAF inhibitors, and others have early resistance development. Pretreatment analysis of BRAFV600 mutation in cell free DNA from serum or plasma could help to select patients for BRAF inhibitor treatments. We have investigated the sensitivity of the assay and its predictive value. Methods: Analysis of BRAFV600 mutation was performed in cell free DNA (cf DNA) from serum and plasma of 22 metastatic melanoma patients before starting a BRAF inhibitor treatment. Results: Six patients were women, median age was 62 years old (58-81). Four patients (18%) had complete response to treatment. Eleven patients (50%) had partial response to treatment. Two patients (9%) had progression disease as their best response. Median progression free survival time (PFS) for patients with complete response, partial response, stable disease and progression disease were: not reached, 4.6 months, 3 months and one month, respectively (p&amp;lt;0.00001). Twelve patients (54.5%) had BRAF positive analysis pretreatment in serum/plasma. From BRAF serum/plasma positive patients 1 had CR (8%) and 7 PR (58%). From BRAF serum/plasma negative patients: 3 (30%) CR and 4 (40%) PR. Median PFS for patients treated with BRAF inhibitors was 3.5 months for BRAF serum/plasma positive patients and 13.57 months for BRAF serum/plasma negative patients (p=0.026) Conclusion: BRAFV600 serum/plasma analysis is a good predictor of survival for patients treated with BRAF inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C41. Citation Format: Maria Gonzalez Cao, Salvador Martin Algarra, Eva Muñoz, Clara Mayo de las Casas, Jose Luis Manzano, Javier Cortes, Miguel Angel Molina-Vila, Jose Pablo Berros, Leticia De Mattos-Arruda, Miguel Sanmamed, Alvaro Gonzalez, Carlos Alvarez, Niki Karachaliou, Rafael Rosell. BRAFV600 serum/plasma analysis: Predictive value of survival in melanoma treated with BRAF inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C41.

Abstract A57: Chondroitin sulfate proteoglycan 4(CSPG4) Mediates Melanoma Cell Survival During PI3K and mTOR Inhibition

Abstract Background: Early development of resistance to therapy remains a major cause of cutaneous malignant melanoma treatment failure. Chondroitin sulfate proteoglycan 4 (CSPG4) is robustly expressed in a majority of metastatic melanomas. Previous work has shown CSPG4 to promote MAPK, PI3K and FAK activation, suggesting that CSPG4 represents a potential avenue for oncogenic growth and cell survival in response to chemotherapy. The PI3K-AKT-mTOR pathway regulates the cell growth and motility in the presence of nutrients. The inhibition of AKT and mTOR independently was shown to reduce the melanoma cell survival and enhanced the sensitivity of these cells to cisplatin. PF-05212384 (PKI-587) is a dual PI3K/mTOR inhibitor in clinical trials. We hereby report that the expression of CSPG4 on the melanoma cells caused them to become less responsive to the therapy with a dual PF-05212384. Material and methods: Melanoma cell lines that stably expressed CSPG4 were selected for this study. The expression of CSPG4 was knocked down by using lentivirus-transduced shRNA. Cell survival assays were performed to compare the inhibitory effects of the drug in CSPG4 expressing and non-expressing cell line. Wound healing migration assays were performed to examine the cell motility and migration. The activation of PI3K-mTOR pathway was analyzed by phosphorylation of downstream effectors for their activation using Western blotting. Results: Melanoma cells expressing active CSPG4 showed less sensitivity to the inhibitor. The average cell viability after treatment with maximum dose was 47.94% and 35.76% for CSPG4 positive 1205LU and WM1341D melanoma cell lines respectively, while the CSPG4 negative WM1552C showed an average cell viability of 19.07%. The difference in cell viability was statistically significant.(p&amp;lt;α=0.05)for higher doses. The suppression of CSPG4 using the shRNA sensitized the CSPG4 positive cell lines to the PI3K/mTOR inhibition. (p&amp;lt;α=0.05). The maximum effect was seen in the WM1341D where the cell viability was reduced at maximum dose to 26.30% as compared to 50% in non-targeting shRNA transduced control. The CSPG4 positive cell lines resisted the deactivation of p70Sk, a downstream effector of mTOR and had enhanced phosphorylation of transcription repressor 4E-BP1.At higher doses the cleavage of caspase to its activated forms, which is necessary for execution of apoptosis was reduced in the CSPG4 positive cell lines. The CSPG4 positive cells showed more motility as compared to the controls. Conclusion: This data supports the hypothesis that the CSPG4 is involved in inducing resistance in melanoma cells in response to the therapeutic challenge with the dual PI3k/mTOR inhibitor. The study highlights the need of using multiple treatment strategies to treat melanoma.

RAB8 Enhances TMEM205-Mediated Cisplatin Resistance

To determine whether the small endosomal recycling GTPase, RAB8, plays a role in TMEM205-associated resistance to the chemotherapeutic drug cisplatin. Antibodies were used as markers for both genes; confocal microscopy was used to visualize their localization in cisplatin-resistant cells. Both single and dual-transfections were performed. Expression of RAB8 was markedly elevated in human cisplatin-resistant cells. We found that TMEM205 was co-localized with RAB8. Dual transfectants with over-expression of both TMEM205 and RAB8 were found to be up to 4-fold more resistant to cisplatin, while cells transfected with RAB8 alone were ~2-fold more resistant. The development of cisplatin resistance appears to be a consequence of pleotropic epigenetic alterations. We unravel the role of one gene, the GTPase RAB8, in this process. Because its highest expression was at an early step of cisplatin resistance, it may be involved in early development of resistance. Increased expression of TMEM205 and RAB8 in double-transfected cells and their increased resistance to cisplatin indicate an additive effect of these two genes, mediating cisplatin resistance. These two proteins are potential biomarkers or targets for gene or chemotherapy.