CT DNA Research Articles

Abstract PD9-06: Paired pre- and post-treatment DNA sequencing identifies genomic alterations mediating clinical resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer

Abstract Background: The combination of cyclin-dependent kinase 4 and 6 inhibitors (CDK 4/6 inh) and endocrine therapy have changed the treatment paradigm for hormone receptor-positive metastatic breast cancer (HR+ MBC). Three CDK 4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) are now approved for HR+ MBC in combination with either an aromatase inhibitor (AI) or fulvestrant (a selective estrogen receptor degrader, SERD) after demonstrating significant improvement in progression-free survival compared to endocrine monotherapy. However, both primary and secondary resistance has been observed clinically, and the mechanisms of such resistance are not well understood. Here we describe emergence of molecular alterations following treatment with the combination of CDK 4/6 inhibitors and endocrine therapy. Methods: We evaluated molecular alterations by next-generation sequencing (NGS) prior to and following treatment with CDK 4/6 + endocrine therapy pre and post CDK inh among patients with HR+ MBC. We performed circulating tumor (ct) DNA analysis via the Guardant360 assay, an NGS-based assay that identifies potential tumor-related (somatic) genomic alterations through sequencing of the critical exons within 73 cancer-related genes, and/or tissue-based genomic analysis utilizing our institutional NGS platform (SNaPshot), which targets hotspots and exons in 89 genes. Results: A total of 33 HR+ MBC patients had paired pre/post CDK 4/6 tissue/liquid biopsies. 19 (57.6%) received palbociclib and 14 (42.4%) ribociclib, with 25 (75%) receiving an AI and 8 (24%) a SERD. Eight patients (24.2%) were first line. The median duration of response was 8 months (range 1-35 months). The most common acquired alterations at the time of progression that were not present in the pre-treatment specimens included mutations in ESR1 (30.3%), TP53 (30.3%), RB1 (12.1%) and PIK3CA (9.1%), and FGFR1 amplification (27.3%). We then evaluated these results in a subset of patients (N=17) who had paired pre/post liquid biopsies only and found similar results. We observed alterations in oncogenic pathways both upstream and downstream of CDK 4/6-cyclin-D complex (N=17), including upstream receptor tyrosine kinase alterations (29.4%), PI3K/AKT/mTOR alterations (5.9%), and MAPK alterations (17.6%), as well as downstream cell-cycle (35.3%) and DNA repair gene alterations (17.6%). Furthermore, 58.8% of patients had more than one acquired potential driver mutation, and 41.2% had both upstream and downstream alterations possibly reflecting emergence of different subclones and tumor heterogeneity under selective pressure from CDK 4/6 inhibitors. Conclusion: Both upstream and downstream genomic alterations may mediate acquired clinical resistance to CDK 4/6 inhibitors, reflecting genomic evolution and diversification after exposure to CDK 4/6 inhibitors. Additional studies are needed to confirm these findings and develop potential therapeutic strategies to overcome clinical resistance to CDK 4/6 inhibitors for patients with MBC. Citation Format: Spring LM, Haddad S, Malvarosa G, Vidula N, Juric D, Iafrate AJ, Ellisen LW, Moy B, Isakoff SJ, Bardia A. Paired pre- and post-treatment DNA sequencing identifies genomic alterations mediating clinical resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-06.

Safety and efficacy of dasatinib in patients with advanced gastrointestinal stromal tumors refractory to imatinib and sunitinib: A single arm, multicenters, phase 2 trial.

138 Background: Regorafenib is recommended to treat advanced gastrointestinal stromal tumor (GIST) refractory to imatinib and sunitinib. However, the efficacy is not satisfied, other active agents need to be explored to advanced patients. Methods: In this single arm, multi-center, phase 2 trial, we enrolled patients aged 18 years and older with advanced GIST who had received previous imatinib and sunitinib treatment. Participants were treated with oral dasatinib 50mg twice a day for 2 weeks. If patients were tolerable, then they received dasatinib 70mg twice a day treatment, to tumor progression or intolerable toxicities. The primary endpoint was RECIST-based progression-free survival (PFS) in the intention-to-treat population. The secondary endpoints included response rate, overall survival (OS) and advent events. ctDNA will be analysis in some patients to explore the sensitive biomarker to dasatinib. Results: From May 2016 to June 2018, 58 patients from nine medical centers were enrolled in this study. Two patients had partial response and disease control rate was 62.0%. The median PFS was 3.0 months (95% CI, 2.6-3.4 months). There was no statistic difference of PFS in both subgroup with different primary mutations and in subgroup with different secondary mutations. The patients with wild type GIST had a trend of longer PFS of 5.5 months. In 4 patients with PDGFRA D842V mutation, two patients had stable disease. The median OS was 14.0 months (95% CI, 10.8-17.2 months). The most frequently observed grade 3 adverse events included anemia (10.3%), diarrhea (1.7%). The analysis of ct DNA is ongoing. Conclusions: Dasatinib is an active treatment for patients with GIST who are refractory to imatinib and sunitnib. This study is registered with ClinicalTrials.gov, NCT02776878 . Clinical trial information: NCT02776878.

A hydroxyquinoline-appended ruthenium(II)-polypyridyl complex that induces and stabilizes G-quadruplex DNA

A polypyridyl ruthenium(II) complex with hydroxyquinoline-derived ligand has been synthesized and characterized. The ability to act as telomeric quadruplex inducer and stabilizer, and quadruplex-binding properties of the complex have been evaluated by absorption and emission analyses, fluorescent intercalator displacement (FID) titrations, circular dichroism (CD) spectroscopy, polymerase chain reaction (PCR) stop assay, color reaction studies, fluorescence resonance energy transfer (FRET) melting assay, Job plot, and molecular modeling. Observations revealed that the complex could well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and showed superior G-quadruplex selectivity over duplex DNA with remarkable ΔTm value of 18.0 °C even at 50-fold excessive supplies of calf thymus (ct) DNA. The complex exhibited high interaction affinity of 2.56 × 106 M−1 with G-quadruplex DNA and evident luminescence enhancements of 3.1 and 4.2 times for quadruplex binding in Na+ and K+ buffer, respectively. In addition, the 1:1 [quadruplex]/[complex] binding mode ratio was determined. The results suggest that the complex can be developed as potential anticancer reagent through binding and stabilization of G-quadruplex DNA.

Structure, DNA/proteins binding, docking and cytotoxicity studies of copper(II) complexes with the first quinolone drug nalidixic acid and 2,2'‑dipyridylamine.

This work presents the synthesis, structural characterization and biological affinity of the newly synthesized copper(II) complexes with the first antibacterial quinolone drug nalidixic acid (nal) or N-donor ligand 2,2'‑dipyridylamine (bipyam). [Cu(II)(nal)(bipyam)Cl], (2) reveals a distorted square pyramidal based geometry in Cu(II) atom confirmed by X-ray crystallography technique. The theoretical stabilities and optimized structures of the complex were obtained from DFT calculations. The ability of the complexes to bind with calf thymus DNA (CT DNA) were investigated by electronic absorption, fluorescence, circular dichroism, and viscosity measurements techniques. The experimental results reveal that the complexes strongly interact with CT DNA via intercalative mode but complex 2 exhibits the highest affinity giving Kb=3.91±0.13×106, M-1. The fluorescence spectroscopy measurements show that both complexes have the superior ability to the replacement of EtBr from DNA-bound EtBr solution and bind to DNA through intercalative mode. Both complex also shows the superior affinity towards proteins with comparatively high binding constant values which have been further revealed by fluorescence spectroscopy measurements. Molecular docking analysis indicates that the interaction of the complexes and proteins are stabilized by hydrogen bonding and hydrophobic interaction. Furthermore, the results of in vitro cytotoxicity reveal that the complex 2 has excellent cytotoxicity than 1 against human breast cancer cell lines (MCF-7).

Synthesis, In Vitro Biological Evaluation and In Silico Studies of Some New Heterocyclic Schiff Bases

Abstract2‐Hydroxy‐naphthaldehyde based heterocyclic Schiff base derivatives (1 a‐1 h) were prepared and characterized by multi‐spectroscopic techniques and elemental analysis. Antibacterial activity of all the compounds was tested against Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae and Escherichia coli bacterial strains. The antifungal potential of the synthesized compounds was also tested against three Candida strains (Candida albicans, Candida glabrata and Candida tropicalis). In the antibacterial activity, the compounds showed high MIC values and thus found less potent against all the tested bacterial strains. Interestingly, compounds 1 b and 1 c exhibited significant activity with MIC 125 μg/ml against all the tested fungal strains. Hemolytic assay against human RBCs revealed that compounds 1 b and 1 c showed less toxicity than the standard drug fluconazole at each tested concentration (25‐1000 μg/ml). In growth kinetics studies, compounds 1 b and 1 c significantly inhibited the growth of Candida cells at 2MIC and MIC concentrations. The interaction ability of lead compounds (1 b and 1 c) with Ct–DNA was carried out by absorption, fluorescence, hydrodynamic, cyclic voltammetery measurements and circular dichroism. Results suggested that compound 1 b and 1 c bind to Ct–DNA via an intercalative mode supported by molecular docking studies. The antioxidant potential of heterocyclic derivatives 1 b and 1 c was estimated by DPPH free radical and hydrogen peroxide assay which confirm that compounds exhibited significant antioxidant activity.

Synthesis and crystal structure of the first dinuclear zinc complex containing 1,4,7-triazacyclononane and biological properties of the protonated ligand

A new binuclear complex, [Zn2L2Cl4]·2H2O {L = N-aldehyde-N-(4-(benzyloxy)benzyl)-1,4,7triazacyclononane}, was synthesized and characterized by X-ray, elemental analysis, infrared and electronic spectroscopy, and mass spectrometry. The central ion is bridged by the L and lies in a tetrahedral configuration with Zn···Zn distance of 6.283 Å. The complex crystallizes in the triclinic space group Pī. ESI-MS of the complex indicates that the protonated ligand HL+ is the active species. The interaction of HL+ with calf thymus–DNA (CT–DNA) and bovine serum albumin (BSA) was studied by means of various spectroscopic methods, which revealed that HL+ could interact with CT–DNA through groove-binding mode and could quench the intrinsic fluorescence of BSA in a static quenching process. DNA–cleavage experiments indicate that HL+ exhibits efficient DNA–cleavage activity in the presence of H2O2, hydroxyl radical (HO•) may serve as the major cleavage active species, and the pseudo-Michaelis–Menten kinetic parameters (Kcat, KM, Vmax); 2.47 h−1, 2.70 × 10−4 M and 6.68 × 10−4 Mh−1.

New multinuclear Scaffold molybdocene-gold lidocaine complex: DNA/HSA binding, molecular docking, cytotoxicity and mechanistic insights

The new heteronuclear molybdocene-gold complex 1, [(η5-Cp)2MoII[(μ2-η2-dtc)2Nap]AuIII(LC)](PF6), (η5-Cp: η5-cyclopentadienyl, (dtc)2Nap: 2,7-bis(dithiocarbamate)naphthalene, LC: lidocaine) was synthesized and evaluated for biological activity. With the aim of assessing the possible DNA-binding mode, the interaction of the complex 1 with calf thymus DNA (CT DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement. Also, the binding of the complex to human serum albumin (HSA) was considered by UV–Vis and fluorescence emission spectroscopy. Moreover, molecular docking was used for modeling of the binding of the complex to DNA and HSA. These experimental results were confirmed by the results of molecular docking concerning the lowest binding energy. The cytotoxicity of the heterometallic complex 1 has been evaluated against a panel of several cancer cell lines with low micromolar IC50 (72 h) values, according to its cellular uptake and also versus HEK293 nonmalignant fibroblasts. Moreover, the complex 1 showed the induction of apoptotic process.Communicated by Ramaswamy H. Sarma

Antiproliferative activity of morpholine-based compounds on MCF-7 breast cancer, colon carcinoma C26, and normal fibroblast NIH-3T3 cell lines and study of their binding affinity to calf thymus-DNA and bovine serum albumin

This report describes the results of a study on the antiproliferative activity of the morpholine-based ligand 1,3-bis(1-morpholinothiocarbonyl)benzene (HL) and its nickel(II) complex (NiL) against human breast cancer cells (MCF-7), colon carcinoma cells (C26), and normal fibroblast NIH-3T3 cells. NiL showed better cytotoxicity on both cancerous cells relative to normal cells in vitro with the highest selective index of 2.22 in MCF-7 cells. The interaction of both compounds with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was studied using various spectroscopic techniques and analytical methods such as UV − vis titrations, thermal denaturation, circular dichroism, competitive fluorescent intercalator displacement assays, as well as molecular modeling. The fluorescence intensity of the probe molecule increases clearly when HL and NiL are added to the methylene blue (MB)–DNA system. Furthermore, the binding of HL and NiL quenches the BSA fluorescence, revealing a 1:1 interaction with a binding constant of about 105 M−1.Communicated by Ramaswamy H. Sarma

New Ionic Cu(II) and Co(II) DACH–Flufenamate Conjugate Complexes: Spectroscopic Characterization, Single X–Ray Studies and Cytotoxic Activity on Human Cancer Cell Lines

AbstractNew ionic antitumor drug entities [{Cu(DACH)2(H2O)Cl}.(fluf)], 1 and [{Co(DACH)2(H2O)Cl}.(fluf)], 2 where DACH=1,2–diamminocyclohexane and fluf=flufenamate anion were prepared and characterized by spectroscopic studies and single X–ray crystallography. In order to evaluate the potential of these complexes to act as antitumor drug candidates, in vitro binding studies of 1 and 2 with ct–DNA have been carried out by biophysical methods which revealed electrostatic binding mode of these drug entities with ct–DNA preferably by external surface or groove binding mode contrary to other non–steroidal anti–inflammatory (NSAIDs) drugs which show intercalative binding. Comparative electron paramagnetic resonance (EPR) titrations were performed which revealed that there was no significant change in EPR spectra of complexes upon incubation with ct–DNA implicating that coordination geometry of metal ions does not alter. Validation of antitumor potential of 1 and 2 was done by cytotoxicity experiments against human cancer cell lines by Sulforhodamine B (SRB) assay. Complex 1 was active against all tested cell lines with an exceptionally low GI50 value=2.9 μg/ml against MCF–7 (breast cancer) cell line showing its preferential selectivity. On the contrary, complex 2 showed poor activity against all tested cancer cell lines.

Tetrazolo[1,5-a]pyrimidine-based metal(II) complexes as therapeutic agents: DNA interaction, targeting topoisomerase I and cyclin-dependent kinase studies

A series of mononuclear nickel(II), copper(II) and zinc(II) complexes of the type [ML1‒3Cl2] (1–9) have been synthesized and characterized. All the complexes interact with CT–DNA through groove mode of binding and bring about prominent nuclease activity on plasmid DNA. The complexes strongly inhibit the topoisomerase I, and also strongly interact with cyclin-dependent kinase receptor. The cytotoxicity activity were assessed aganist three human cancer cell lines such as lung (A549), cervical (HeLa) and colon (HCT–15), in which the complex 5 shows highest activity against the colon (HCT-15) cancer cell. The apoptotic studies were also reported.

Obinutuzumab Plus Gemcitabine, Dexamethasone and Cisplatin (O-GDP) As Salvage Chemotherapy Prior to Autologous Stem Cell Transplant in Aggressive B Cell Lymphoma

Background: The LY.12 randomized phase 3 clinical trial defined gemcitabine, dexamethasone and cisplatin (GDP) as an effective outpatient salvage chemotherapy regimen in relapsed/refractory (R/R) patients with aggressive lymphomas who are candidates for autologous stem cell transplant (ASCT) (Crump et al. JCO 2014). When the anti-CD20 antibody rituximab (R) was added to GDP, the ORR was 45.6% by CT imaging and 51.9% of patients were able to receive ASCT. Obinutuzumab (O) is a type 2 CD20 antibody that has demonstrated superiority to R in some studies in indolent lymphomas and is active in R-refractory follicular lymphoma. Improvements in the outcome of salvage therapy have tested alternative CD20 antibodies (Van Imhoff, JCO 2017), to date without success. We report a single centre, single arm clinical trial of O-GDP to assess safety and efficacy in R/R aggressive B cell lymphoma.Methods: Transplant eligible patients with DLBCL and transformed indolent lymphoma were treated with O-GDP for two cycles, followed by response assessment by CT. Non-progressors received a third cycle of O-GDP for stem cell mobilization and a PET/CT scan was obtained after stem cell collection. Responders then proceeded to ASCT per investigator decision. O was given at 1000 mg weekly during the first cycle of GDP and then on day 1 of cycles 2 and 3. Responses were determined by Lugano criteria using investigator assessment. The primary outcome was ORR by CT imaging after 2 cycles. The pre-specified statistical analysis stated that the trial will be declared positive if the ORR was >60%, negative if the ORR was <40% and indeterminate if in-between. Secondary outcomes included PET CR rate after 3 cycles of O-GDP and the rate of proceeding to ASCT. Exploratory outcomes included measurements of circulating tumor (ct) DNA during protocol therapy, and analysis of individual mutations.Results: The trial has completed its planned accrual of 30 patients. Median age was 59.5 (range 30 - 70), with 40% female, 70% with DLBCL NOS and 30% with transformed indolent lymphoma (all but one patient having transformed follicular lymphoma). IPI at study entry was 0-1 (20% of patients), 2 (30%) or 3+ (50%). Grade 3 or 4 toxicity was observed in 87% of patients. This was mainly myelosuppression with grade 4 neutropenia (47%) and thrombocytopenia (37%). Other grade 4 adverse events were sepsis (2 patients), febrile neutropenia (1 patient) and hypokalemia (1 patient). No grade 5 events were attributed to study treatment. One additional case of myelodysplastic syndrome caused treatment discontinuation. Events necessitating dose reductions (33% of patients), dose delays (23%) and dose holds (23%) occurred in a total of 57% of patients.At the time of submission, data are evaluable for the primary endpoint in 28 patients. The ORR (CR + PR) by CT imaging after 2 cycles of study therapy was 60.7% (95% CI 40.6-78.5) Overall, 65.5% of patients (95%CI = 45.7%-82.1%) proceeded to ASCT. In addition, 15 patients are evaluable by PET/CT after 3 cycles of O-GDP, with 47% attaining a complete metabolic response (Deauville 1 - 3), 40% a partial metabolic response and 13% with no metabolic response by Lugano criteria. Plasma ctDNA measurements were taken at baseline, after 1 cycle of O-GDP, and at the time of PET/CT. CtDNA quantities will be compared to imaging assessments.Conclusions: O-GDP is an outpatient salvage regimen that enables ASCT in patients with R/R DLBCL. Compared to R-GDP there is a greater frequency of grade 3 - 4 toxicity (O-GDP 87%, R-GDP previously reported at 47%), although the difference is mainly due to cytopenias and can be managed by dose adjustments to the GDP regimen. Further data analysis is required to determine if the trial will meet the primary endpoint of an ORR > 60%. The overall rate of proceeding to ASCT with O-GDP salvage in this trial was 65.5% compared to that previously reported for R-GDP at 51.9%. DisclosuresScott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Consultancy, Honoraria. Kuruvilla:BMS: Consultancy, Honoraria; Abbvie: Consultancy; Leukemia and Lymphoma Society Canada: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Princess Margaret Cancer Foundation: Research Funding; Gilead: Consultancy, Honoraria; Lundbeck: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

Purpose: The accumulation of emergent RAS mutations during anti-EGFR therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status.Experimental Design: This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the PlasmaSelect-R 64-gene panel at 0.1% sensitivity.Results: Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval, 11.5-15.4) and 7.9 months (6.4-9.6), respectively (P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations.Conclusions: Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Clin Cancer Res; 24(22); 5602-9. ©2018 AACR.

Molecular detection of Chlamydia trachomatis infection among males with abnormal semen

Many studies revealed that Chlamydia trachomatis is the most prevalent bacteria “that cause sexually transmitted infections in the world.. Most of these infections are asymptomatic and there is a remarkable relationship between CT infections and male infertility. Therefore, “the present study is established to determine the effect of Chlamydia trachomatis infection on seminal fluid quality in males suffering from infertility compared with fertile males.. “Semen samples were collected from 63 infertile male and 13 fertile male as control group, attending the clinical laboratory for routine seminal fluid analysis. Seminal fluid was analyzed according to World Health Organization guidelines, the whole genomic DNA was extracted for molecular study. Real time PCR technique was used to specifically detect the presence of CT DNA in semen samples.” trachomatis was demonstrated in the semen samples of 11 (17.4%) infertile male and all control samples were negative. Infertile, infected males had semen samples that showed statistically significant differences in the mean of total sperm count, motility and morphology as compared with fertile uninfected control samples. These differences indicate that chlamydial infection of genital tract could negatively influence the quality of seminal fluid.”

Carbonyl–heterobimetallic Ru(II)/Fe(II)–complexes containing polypyridyl ligands: Synthesis, characterization, cellular viability assays and interactions with biomolecules

This paper describes on the interaction studies of carbonyl heterobimetallic compounds of Ru(II)/Fe(II) containing polypyridyl ligands, with general formula ct–[RuCl(CO)(N–N)(dppf)]PF6, N–N = 1,10–phenanthroline (phen) 5; dipyrido[3,2–f:2′,3′–h]quinoxaline (dpq) 6; dipyrido[3,2–a:2′,3′–c]phenazine (dppz) 7; dipyrido[3,2–f:2′,3′–h]quinoxalino[2,3–b]quinoxaline (dpqQX) 8 and dppf = 1,1‘–bis(diphenylphosphino) ferrocene], with calf thymus DNA (ct–DNA) and bovine serum albumin (BSA). Also, it describes the cellular viability assays of these complexes in tumorigenic and non-tumorigenic cell lines. The carbonyl complexes 5–8 and their respective precursors with formula cis–[RuCl2(N–N)(dppf)], N–N = phen (1), dpq (2), dppz (3) and dpqQX (4), were characterized by elemental analysis and spectroscopic techniques (FTIR, UV–vis, 1H and 31P{1H} NMR). Also, a cyclic voltammetry study was performed for all complexes. The crystal structure of the complex 3 is presented and discussed. Spectrofluorimetric titrations shows spontaneous and strong interaction of 5–8 with BSA, through a static quenching mechanism, resulting in binding constants in the order of 104–106 L mol−1, at 310 K. Viscosity measurements and circular dichroism spectra prompts interactions of 5–8 with ct–DNA via non–classical intercalations or by an electrostatic pathway. MTT assays in breast tumor cells MDA–MB–231 and in non-tumorigenic cells MCF-10A and V79–4 cell lines revealed IC50 values ranging from 0.19 to 1.11 μmol L−1, 1.07–3.18 μmol L−1 and 1.29–3.85 μmol L−1 respectively, for complexes 5–8.

Synthesis, biological evaluation and molecular docking studies on the DNA and BSA binding interactions of palladium(II) and platinum(II) complexes featuring amides of tetrazol-1-yl- and tetrazol-5-ylacetic acids

Novel palladium(II) and platinum(II) complexes (trans-[PdCl2L2] and trans-[PtCl2L2] {L = 2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-(sec-butyl)-2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-cyclopropyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-cyclohexyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide, 2-(2-(tert-butyl)-2H-tetrazol-5-yl)acetamide and 2-(2-(tert-butyl)-2H-tetrazol-5-yl)-N-cyclohexylacetamide} 1b–6b and 1c–4c) were synthesized and characterized by elemental analyses (CHN), HRESI+-MS, 1H, 13C{1H}, 195Pt NMR and IR spectroscopies, DSC/TG analysis, as well as by X-ray single crystal diffraction {for (2-(5-methyl-1H-tetrazol-1-yl)acetamide) 1a·H2O, trans-[PdCl2(2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 1b, trans-[PdCl2(N-cyclopropyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 3b and trans-[PdCl2(N-cyclohexyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 4b}. The binding of complexes 2b and 5b to calf-thymus DNA (CT DNA) and BSA (bovine serum albumin) was studied by means of CD, UV and fluorometric techniques. According to the spectroscopic data, an interaction of the metal complexes with CT DNA was confirmed. A significant increase in the melting point of CT DNA in the presence of the metal complexes {ΔTm = 10.0 °C (for 2b), 12.5 °C (for 5b)} indicates a strong stabilization of the DNA helix. According to the fluorometric data, complex 2b exhibits an interaction with BSA. Additionally, molecular docking results suggest that complex 1b and its fully hydrolyzed form (trans-[Pd(H2O)2(2-(5-methyl-1H-tetrazol-1-yl)acetamide)2]) cooperatively interact with DNA, presumably via minor groove binding. The hydrolyzed form possesses a greater affinity to DNA. Also, there is an effective interaction of complex 1b and the corresponding hydrolyzed form to BSA via the two binding sites Trp134 and Trp213. Two complexes {trans-[PdCl2(N-(sec-butyl)-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 2b and trans-[PdCl2(2-(2-(tert-butyl)-2H-tetrazol-5-yl)acetamide)2] 5b} demonstrate antiproliferative activity against human cancer cell lines, with IC50 = 45 ± 0.5 µM for 2b on HeLa and IC50 = 44.3 ± 0.8 µM for 5b on MCF-7. The complexes 4b, 4c and 6b were evaluated for their antimicrobial activity against Escherichia coli (strain КA796) and they showed significant activities, being greater than that of cisplatin. Complex 4b was the most effective, with the lowest MLC value of 0.25 µM and LC50 value of 0.075 µM.

Synthesis and DNA binding profile of monomeric, dimeric, and trimeric derivatives of crystal violet

Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a–1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a–1f have apparent association constants for CT DNA in the range of 1.80–16.2 × 107 M−1 and binding site sizes of 10–14 bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove.

Studies on the interaction of mononuclear metal(II) complexes of amino‑naphthoquinone with bio-macromolecules

Three metal(II) complexes [CoLCl2], [CuLCl2] and [ZnL2Cl2] {L = 2‑chloro‑3‑((3‑dimethylamino)propylamino)naphthalene‑1,4‑dione} have been synthesized and characterized using analytical, thermal and spectral techniques (FT-IR, UV–Vis, ESR and ESI-MS). The structure of the L has been confirmed by single crystal XRD study. The complexes show good binding propensity to bovine serum albumin (BSA) having relatively higher binding constant values (104 M−1) than the ligand. Fluorescence spectral studies indicate that [CoLCl2] binds relatively stronger with CT DNA through intercalative mode, exhibiting higher binding constant (2.22 × 105 M−1). Agarose gel electrophoresis run on plasmid DNA (pUC18) prove that all the complexes showed efficient DNA cleavage via hydroxyl radical mechanism. The complexes were identified as potent anticancer agents against two human cancer cell lines (MCF7 and A549) by comparing with cisplatin. Co(II) complex demonstrated greater cytotoxicity against MCF7 and A549 cells with IC50 values at 19 and 22 μM, respectively.

LBA55 - Primary efficacy results from B-F1RST, a prospective phase II trial evaluating blood-based tumour mutational burden (bTMB) as a predictive biomarker for atezolizumab (atezo) in 1L non-small cell lung cancer (NSCLC)

Background: Blood and tissue TMB have shown promise in predicting benefit from PD-L1/PD-1 inhibitors. High bTMB enriched for a PFS benefit in patients (pts) treated with atezo monotherapy in retrospective analyses from the randomized, 2L NSCLC Ph III OAK and Ph II POPLAR studies. We report primary results from the fully enrolled prospective, Ph II, B-F1RST study evaluating a novel bTMB assay as a predictive biomarker for atezo in 1L NSCLC. Methods: Of 152 ITT pts, 119 comprised the biomarker-evaluable population (BEP) that had adequate circulating tumor (ct)DNA (maximum somatic allele frequency [MSAF] ≥ 1%); 29 had inadequate ctDNA (non-BEP [MSAF <1%]). A prespecified bTMB cutoff (high ≥ 16; low < 16) was used to evaluate clinical efficacy. Coprimary endpoints were ORR and PFS. Statistical tests were 2-sided at a 0.1 level. Results: With minimum follow-up of ≥ 6 mo, confirmed ITT ORR was 14.5% (22/152). ORR in BEP was 10.1% (12/119) and non-BEP was 34.5% (10/29). In bTMB high vs low pts, ORR was 28.6% (8/28) vs 4.4% (4/91) and mPFS was 4.6 vs 3.7 mo; HR = 0.66 (90% CI, 0.42–1.02; P = 0.12). mPFS at exploratory bTMB cutoffs are presented in the Table. bTMB high vs low mOS was NE vs 13.1 mo; HR = 0.77 (90% CI, 0.41–1.43; P = 0.48). 13% of pts had treatment-related serious AEs and 20% had treatment-related Gr 3/4 AEs. 15% of pts had AEs leading to discontinuation. Conclusions: The B-F1RST primary analysis is the first full prospective dataset evaluating clinical utility of bTMB as a predictive biomarker for 1L pts receiving atezo monotherapy. Consistent with interim data, pts at the prespecified bTMB ≥16 cut-off had numerical benefit for PFS, ORR and OS. Pts will be followed for ≥18 mo per protocol. Additional exploratory analyses on the bTMB biomarker will be reported.Table: LBA55PFS (mo) by bTMB scores (BEP, n = 119)bTMB Low (< bTMB cutoff)bTMB High (≥ bTMB cutoff)bTMB cutoffmedian (n)median (n)HR90% CI124.1 (75)2.6 (44)1.010.70, 1.46144.1 (84)2.6 (35)0.920.62, 1.37163.7 (91)4.6 (28)0.660.42, 1.02183.2 (96)6.9 (23)0.460.28, 0.76202.9 (100)6.9 (19)0.480.28, 0.82 Open table in a new tab Clinical trial identification: NCT02848651. Editorial acknowledgement: Christopher Lum, PhD, Health Interactions. Legal entity responsible for the study: F. Hoffmann-La Roche AG. Funding: F. Hoffmann-La Roche AG. Disclosure: E.S. Kim: Consulting, advisory role: Lilly, Celgene, AstraZeneca, Boehringer Ingelheim; Travel, accommodations, expenses: Lilly, Celgene, AstraZeneca, Boehringer Ingelheim; Honoraria: Celgene, Lilly, AstraZeneca, Boehringer Ingelheim. V. Velcheti: Speakers bureau, advisory board: Genentech, BMS, Celgene, AstraZeneca, Takeda Oncology, Foundation Medicine. T. Mekhail: Speakers bureau: Genentech/Roche, Lilly, Bristol-Myers Squibb, Merck; Honoraria: Genentech/Roche, Lilly, Bristol-Myers Squibb, Merck, Boehringer Ingelheim. T.A. Leal: Consulting, advisory board: Takeda, AstraZeneca, Novartis, AbbVie, BMS. V. Shen: Employee of Roche/Genentech. S. Hu, S.M. Paul: Employee of Genentech Inc., a member of the Roche Group. D.S. Shames: Employee of Genentech Inc., a member of the Roche Group, and owns Roche stock. E. Schleifman: Employee and holds stock of Genentech Inc., a member of the Roche Group. D.A. Fabrizio: Employee and stockholder of Foundation Medicine, Inc. C. Yun: Stock or other ownership: Roche; Employment: Genentech; Research funding: Genentech/Roche; Travel, accommodations, expenses: Genentech/Roche. S. Phan: Employee of Genentech Inc., a member of the Roche Group, owns Roche stock and other ownership interests. M.A. Socinski: Honoraria, speakers bureau, research funding: Genentech. All other authors have declared no conflicts of interest.

The diagnostic accuracy of circulating tumor DNA for the detection of EGFR-T790M mutation in NSCLC: a systematic review and meta-analysis

This pooled analysis aims at evaluating the diagnostic accuracy of circulating tumor (ct) DNA for the detection of EGFR-T790M mutation in NSCLC patients who progressed after EGFR-TKIs. Data from all published studies, reporting both sensitivity and specificity of plasma-based EGFR-T790M mutation testing by ctDNA were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference of Lung Cancer meeting proceedings. A total of twenty-one studies, with 1639 patients, were eligible. The pooled sensitivity of ctDNA analysis was 0.67 (95% CI: 0.64–0.70) and the pooled specificity was 0.80 (95% CI: 0.77–0.83). The pooled positive predictive value (PPV) was 0.85 (95% CI: 0.82–0.87) and the pooled negative predictive value (NPV) was 0.60 (95% CI: 0.56–0.63). The positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 2.67 (95% CI: 1.86–3.82) and 0.46 (95% CI: 0.38–0.54), respectively. The pooled diagnostic odds ratio (DOR) was 7.27 (4.39–12.05) and the area under the curve (AUC) of the summary receiver operating characteristics (sROC) curve was 0.77. The ctDNA analysis represents a promising, non-invasive approach to detect and monitor the T790M mutation status in NSCLC patients. Development of standardized methodologies and clinical validation are recommended.

Abstract B32: RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer

Abstract Background: EGFR-TKIs are the recommended first-line therapy of patients (pts) with advanced non-small cell lung cancer (NSCLC) who have an EGFR-sensitizing mutation (EGFRm). However, most pts develop resistance to EGFR-TKIs, with the EGFR T790M resistance mutation observed in &amp;gt;50% of pts on first- and second-generation EGFR-TKIs. Osimertinib is a third-generation, CNS-active EGFR-TKI that potently and selectively inhibits both EGFRm and T790M resistance mutations, recommended for pts with T790M-positive advanced NSCLC who have progressed on first-line EGFR-TKI therapy. Tumor tissue is the preferred sample source for T790M testing; however, often tumor biopsy is not feasible following disease progression due to inaccessible sites and/or concerns about safety. Testing using plasma samples offers a less invasive, albeit less sensitive, alternative method for the detection of T790M-positive circulating tumor (ct) DNA. Additional noninvasive options, including urine ctDNA analyses, are also being investigated. The RADIANCE trial is designed to determine if urine and plasma tests can provide similar detection rates to tissue testing for T790M detection when combined. Pts identified as T790M-positive may be offered treatment with osimertinib. Trial Design: RADIANCE (NCT03137264) is an open-label, prospective biomarker study with a primary objective of assessing the analytic concordance between noninvasive testing (Guardant360 Plasma Assay and Trovera Liquid Biopsy Test using urine) and tissue testing (cobas® EGFR Mutation Test v2) for T790M in pts with NSCLC. Approximately 470 pts will be enrolled. The study consists of two parts, Diagnostic Analytic Validity (Part 1) and Clinical Outcomes (Part 2). In Part 1, eligible pts will provide a tumor biopsy (cobas tissue test), plasma (cobas plasma and Guardant360 tests) and urine sample (Trovera test) for T790M testing. Eligibility criteria include a primary diagnosis of EGFRm-positive NSCLC with evidence of disease progression during or following treatment with a first- or second-generation EGFR-TKI. Pts must not be enrolled on another clinical trial, or have previously received osimertinib or another T790M-directed therapy. In Part 2, pts with T790M-positive status by cobas tissue and/or cobas plasma test will be treated according to standard of care, and may receive osimertinib. Pts will be followed for clinical outcomes for up to 18 months, and evaluated for objective response, progression-free survival, and duration of response (investigator-assessed using RECIST v1.1) and safety. Pts who are T790M negative or do not receive osimertinib will have completed the study after Part 1. Citation Format: Hatim Husain, Martin Dietrich, Steven Dubinett, David E. Gerber, Jeffrey Gregg, Michelle Shiller, Howard West, Kim Thai, Alvin Milner, Nabil Chehab, Christina Baik. RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B32.