Quantification of Biologically Active DNA Alkylation in Temozolomide-Exposed Glioblastoma Cell Lines by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry: Method Development and Recommendations for Validation.

Quantitative monitoring of biologically active methylations of guanines in samples exposed to temozolomide (TMZ) would be useful in glioblastoma research for preclinical TMZ experiments, for clinical pharmacology questions regarding appropriate exposure, and ultimately for precision oncology. The known biologically active alkylation of DNA induced by TMZ takes place on O6 position of guanines. However, when developing mass spectrometric (MS) assays, the possible signal overlap of O6-methyl-2'-deoxyguanosine (O6-m2dGO) with other methylated 2'-deoxyguanosine species in DNA and methylated guanosines in RNA must be considered. Liquid chromatography-tandem MS (LC-MS/MS) offers the analytical requirements for such assays in terms of specificity and sensitivity, especially when multiple reaction monitoring (MRM) is available. In preclinical research, cancer cell lines are still the gold standard model for in vitro drug screening. Here, we present the development of ultra-performance LC-MRM-MS assays for the quantification of O6-m2dGO in a TMZ-treated glioblastoma cell line. Furthermore, we propose adapted parameters for method validation relevant to the quantification of drug-induced DNA modifications.

Important Requirements for Desorption/Ionization Mass Spectrometric Measurements of Temozolomide-Induced 2'-Deoxyguanosine Methylations in DNA.

In clinical pharmacology, drug quantification is mainly performed from the circulation for pharmacokinetic purposes. Finely monitoring the chemical effect of drugs at their chemical sites of action for pharmacodynamics would have a major impact in several contexts of personalized medicine. Monitoring appropriate drug exposure is particularly challenging for alkylating drugs such as temozolomide (TMZ) because there is no flow equilibrium that would allow reliable conclusions to be drawn about the alkylation of the target site from plasma concentrations. During the treatment of glioblastoma, it appears, therefore, promising to directly monitor the alkylating effect of TMZ rather than plasma exposure, ideally at the site of action. Mass spectrometry (MS) is a method of choice for the quantification of methylated guanines and, more specifically, of O6-methylguanines as a marker of TMZ exposure at the site of action. Depending on the chosen strategy to analyze modified purines and 2'-deoxynucleosides, the analysis of methylated guanines and 2'-deoxyguanosines is prone to important artefacts due to the overlap between masses of (i) guanines from DNA and RNA, and (ii) different methylated species of guanines. Therefore, the specific analysis of O6-methyl-2'deoxyguanosine, which is the product of the TMZ effect, is highly challenging. In this work, we report observations from matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray ionization (DESI) MS analyses. These allow for the construction of a decision tree to initiate studies using desorption/ionization MS for the analysis of 2'-deoxyguanosine methylations induced by TMZ.

High expression of the phosphoinositide 3-kinase p11γ isoform can predict poor prognosis of non-small cell lung cancer.

The protein p110γ is an isoform of the catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI3Ks are involved in the regulation of cell survival, growth, proliferation, and migration and have been implicated in the oncogenesis of various cancers. In this study, p110γ expression in non-small cell lung cancer (NSCLC) and its association with clinicopathological factors and patient survival were evaluated. A total of 230 NSCLC tumors were immunohistochemically stained for p110γ. Of these, 174 (75.7%) and 56 (24.3%) were placed in the low and high expression groups, respectively. The positive rate of p110γ was significantly higher in adenocarcinoma than in squamous cell carcinoma (p⟨0.001). Advanced stage NSCLCs showed higher p110γ expression than those at an early stage (p=0.002). Irrespective of the histological tumor type, the patients with high p110γ expression had significantly worse overall survival than those with low p110γ expression (p=0.004). p110γ expression was an independent poor prognostic factor in the multivariate analysis. Our results suggest that p110γ may be involved in the development and progression of NSCLC, and that p110γ has promising potential as a prognostic factor or novel therapeutic target for NSCLC.

Efficacy and safety of Viscum album extract (Helixor-M) to treat malignant pleural effusion in patients with lung cancer.

PURPOSE: Manifestations of malignant pleural effusions (MPEs) are alleviated by local therapies as well as by systemic treatment. After 2009, when commercial use of talc was discontinued in Korea, we have used Helixor-M, which is derived from the European mistletoe (Viscum album), as an alternative sclerosing agent for pleurodesis. We aimed to evaluate the efficacy and safety of Helixor-M for controlling MPE. METHODS: Between 2009 and 2015, we consecutively enrolled 52 patients with lung cancer, who underwent pleurodesis to treat MPE and were analyzed retrospectively. On day 1, 100 mg of Helixor-M was instilled via pleural catheter. If the procedure was not effective, it was repeated every other day up to five times, and the dose increased each time by 100 mg. The primary study outcome was reappearance of pleural effusion at 1 month after the last pleurodesis procedure. RESULTS: The median age of patient was 63 years, and 77% of the 52 patients were male. About 85% of pleural effusions were found to be malignant by cytogenetic analysis. Forty-two (81%) patients were evaluable for recurrence of MPE. The 1-month recurrence rate was 48% (20/42). Among the 20 patients who developed recurrent MPE, 6 required therapeutic thoracentesis. Thirteen (25%) patients experienced procedure-related pain requiring medication. Eight (15%) had fever > 38 °C. CONCLUSIONS: Our results suggest that a pleurodesis with Helixor-M was an effective and tolerable procedure for controlling MPE in lung cancer patients.

Tandem Rh-Catalyzed Oxidative C-H Olefination and Cyclization of Enantiomerically Enriched Benzo-1,3-Sulfamidates: Stereoselective Synthesis of trans-1,3-Disubstituted Isoindolines.

A tandem process, involving Rh(III)-catalyzed oxidative C-H olefination of enantiomerically enriched 4-aryl-benzo-1,3-sulfamidates and subsequent intramolecular aza-Michael cyclization has been developed. The reaction produces trans-benzosulfamidate-fused-1,3-disubstituted isoindolines as major products, in which the configurational integrity of the stereogenic center in the starting material is preserved. Further transformations of the benzosulfamidate-fused-1,3-disubstituted isoindolines are described.

Stereoselective Synthesis of Functionalized 1,3-Disubstituted Isoindolines via Rh(III)-Catalyzed Tandem Oxidative Olefination-Cyclization of 4-Aryl-cyclic Sulfamidate-5-Carboxylates.

A new method for the direct, stereoselective synthesis of highly functionalized 1,3-disubstituted isoindolines 6 from enantiomerically enriched cyclic 4-aryl-sulfamidate-5-carboxylates (5) is described. The process involves sulfamidate directed, Rh(III)-catalyzed tandem ortho C-H olefination of the 4-aryl-sulfamidate-5-carboxylates and subsequent cyclization by aza-Michael addition. In the reaction, which generates trans-1,3-disubstituted isoindolines exclusively, the configurational integrity of the stereogenic center in the starting cyclic sulfamidate is completely retained in the product. Examples are provided which show that the cyclic sulfamidate moiety not only serves as a chiral directing group but also as a versatile handle for further functionalization of the generated isoindoline ring system.

Pharmacological treatment response according to the severity of symptoms in patients with chronic obstructive pulmonary disease.

BACKGROUND: Pharmacological management of chronic obstructive pulmonary disease (COPD) is recommended according to the individualized assessment of symptoms and exacerbation risks. The aim of this study was to determine the relationship between the baseline Modified British Medical Research Council (mMRC) dyspnea scale and the COPD Assessment Test (CAT) score and pharmacological treatment response in patients with COPD. METHODS: A total of 102 stable COPD patients who were enrolled in prospective cohort studies were analyzed. Pharmacological treatment responses after a 3-month treatment were assessed by changes on the mMRC dyspnea scale, CAT scores, and spirometric pulmonary functions. RESULTS: Sixty-two patients with a mMRC dyspnea scale ≤1 were classified as having "less dyspnea" and 40 patients with a mMRC dyspnea scale ≥2 as having "more dyspnea". After a 3-month treatment, the mean mMRC dyspnea scale in the "more dyspnea" group was significantly decreased versus the "less dyspnea" group; however, there were no significant differences in CAT score changes or spirometric pulmonary function changes between the two groups. Baseline mMRC scales (Spearman's rho =-0.591, P<0.001) and baseline CAT scores (Pearson's r =-0.337, P=0.001) were significantly correlated with their changes after a 3-month treatment. Multiple logistic regression analysis demonstrated that baseline mMRC scale and CAT score were the only independent predictors of improvement greater than a minimal clinically significant difference after treatment. CONCLUSIONS: The severity of COPD symptoms is associated with their response to pharmacotherapy. COPD patients with a higher baseline mMRC dyspnea scale and CAT score experience greater symptom reduction by pharmacotherapy.