A global phase II randomized trial comparing oral taxane ModraDoc006/r to intravenous docetaxel in metastatic castration resistant prostate cancer.

STUDY AIM: ModraDoc006, an oral formulation of docetaxel, is co-administered with the cytochrome P450-3A4 and P-glycoprotein inhibitor, ritonavir (r): ModraDoc006/r. The preliminary efficacy and safety of oral ModraDoc006/r was evaluated in a global randomized phase II trial and compared to the current standard chemotherapy regimen of intravenous (i.v.) docetaxel and prednisone. METHODS: 103 mCRPC patients, chemotherapy-naïve with/without abiraterone and/or enzalutamide pretreated, with adequate organ function and evaluable disease per RECIST v1.1 and PCWG3 guidelines were randomized 1:1 into two cohorts. In Cohort 1, 49 patients received docetaxel 75 mg/m2 i.v. every 3 weeks (Q3W). In Cohort 2, 52 patients received ModraDoc006/r; 21 patients with a starting dose of ModraDoc006 30 mg with ritonavir 200 mg in the morning and ModraDoc006 20 mg with ritonavir 100 mg in the evening (30-20/200-100 mg) bi-daily-once-weekly (BIDW) on Days 1, 8, and 15 of a 21-day cycle. To alleviate tolerability, the starting dose was amended to ModraDoc006/r 20-20/200-100 mg in another 31 patients. All patients received prednisone 10 mg daily. Primary endpoint was rPFS. RESULTS: There was no significant difference in rPFS between the 2 arms (p = 0.1465). Median rPFS was 9.5 months and 11.1 months (95% CI) for ModraDoc006/r and i.v. docetaxel, respectively. Partial response was noted in 44.1% and 38.7% measurable disease patients, and 50% decline of PSA was seen in 23 (50%) and 26 (56.5%) evaluable cases treated with ModraDoc006/r and i.v. docetaxel, respectively. The safety profile of ModraDoc006/r 20-20/200-100 mg dose was significantly better than i.v. docetaxel, with mild (mostly Grade 1) gastrointestinal toxicities, no hematologic adverse events, and neuropathy and alopecia incidence of 11.5% and 25%, respectively. CONCLUSIONS: ModraDoc006/r potentially represents a widely applicable, convenient, effective, and better tolerated oral taxane therapy option for mCRPC. Further investigation of ModraDoc006/r in a large randomized trial is warranted.

Clinical predictors of response to single­agent immune checkpoint inhibitors in chemotherapy­pretreated non­small cell lung cancer.

Single-agent immune checkpoint inhibitors (ICIs) are the standard option for chemotherapy-pretreated metastatic non-small cell lung cancer (NSCLC), however only a subset of patients responds to this treatment. The present study aimed at the development of a tool for personalized prediction of the efficacy of ICIs. The study included 181 epidermal growth factor receptor/anaplastic lymphoma kinase-negative patients with metastatic NSCLC receiving single-agent ICI in the second or later line of therapy. For the comparison, a total of 63 metastatic patients with NSCLC treated by chemotherapy were also analyzed. Multivariate analysis revealed that Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2, never-smoking status and the baseline neutrophil-to-lymphocyte ratio (NLR) ≥4.3 were associated with reduced progression-free survival (PFS) and overall survival (OS) [ECOG PS: Hazard ratio (HR)=2.09; P=0.028 and HR=2.02; P=0.035, respectively; never-smoking: HR=3.53; P=0.007 and HR=1.80; P=0.004, respectively; NLR ≥4.3: HR=4.34; P<0.0001 and HR=4.89; P<0.0001 respectively]. Patients with an NLR <4.3, who had a favorable ECOG PS (0-1) and smoking history in the past, derived the utmost benefit from ICI [n=77; objective response rate (ORR)=35%; PFS and OS: 17.1 and 33.7 months, respectively]. The worst efficacy of ICI was observed in patients who had an NLR ≥4.3 coupled with poor ECOG PS and/or never-smoking status (n=38; ORR=8%; PFS=3.2 months and OS=7.2 months). The remaining patients belonged to the group with intermediate outcomes (n=66; ORR=17%; PFS and OS: 4.3 and 12.2 months, respectively). While combination of these factors was highly predictive for ICIs, it was not associated with outcomes of chemotherapy treatment. Easily available characteristics of the patients allow for highly accurate predictions of outcomes of single-agent ICI therapy in chemotherapy-pretreated NSCLC.

Ethnicity-specific BRCA1, BRCA2, PALB2, and ATM pathogenic alleles in breast and ovarian cancer patients from the North Caucasus.

BACKGROUND: Mountain areas of the North Caucasus host several large ethnic communities that have preserved their national identity over the centuries. METHODS: This study involved high-grade serous ovarian cancer (HGSOC) and breast cancer (BC) patients from Dagestan (HGSOC: 37; BC: 198), Kabardino-Balkaria (HGSOC: 68; BC: 155), North Ossetia (HGSOC: 51; BC: 104), Chechnya (HGSOC: 68; BC: 79), Ingushetia (HGSOC: 19; BC: 103), Karachay-Cherkessia (HGSOC: 13; BC: 47), and several Armenian settlements (HGSOC: 16; BC: 101). The group of BC patients was enriched by young-onset and/or family history-positive and/or bilateral and/or receptor triple-negative cases. The entire coding region of BRCA1, BRCA2, PALB2, and ATM genes was analyzed by next-generation sequencing. RESULTS: A significant contribution of BRCA1/2 pathogenic variants (PVs) to HGSOC and BC development was observed across all North Caucasus regions (HGSOC: 19-39%; BC: 6-13%). Founder alleles were identified in all ethnic groups studied, e.g., BRCA1 c.3629_3630delAG in Chechens, BRCA2 c.6341delC in North Ossetians, BRCA2 c.5351dupA in Ingush, and BRCA1 c.2907_2910delTAAA in Karachays. Some BRCA1/2 alleles, particularly BRCA2 c.9895C > T, were shared by several nationalities. ATM PVs were detected in 14 patients, with c.1673delG and c.8876_8879delACTG alleles occurring twice each. PALB2 heterozygosity was observed in 5 subjects, with one variant seen in 2 unrelated women. CONCLUSION: This study adds to the evidence for the global-wide contribution of BRCA1/2 genes to HGSOC and BC morbidity, although the spectrum of their PVs is a subject of ethnicity-specific variations. The data on founder BRCA1/2 alleles may be considered when adjusting the BRCA1/2 testing procedure to the ethnic origin of patients.

Insulin Downregulates the Expression of ATP-binding Cassette Transporter A-I in Human Hepatoma Cell Line HepG2 in a FOXO1 and LXR Dependent Manner.

ATP-binding cassette transporter A-I (ABCA1) is an ubiquitously expressed protein whose main function is the transmembrane transport of cholesterol and phospholipids. Synthesis of ABCA1 protein in liver is necessary for high-density lipoprotein (HDL) formation in mammals. Thus, the mechanism of ABCA1 gene expression regulation in hepatocytes are of critical importance. Recently, we have found the insulin-dependent downregulation of other key player in the HDL formation-apolipoprotein A-I gene (J. Cell. Biochem., 2017, 118:382-396). Nothing is known about the role of insulin in the regulation of ABCA1 gene. Here we show for the first time that insulin decreases the mRNA and protein levels of ABCA1 in human hepatoma cell line HepG2. PI3K, p38, MEK1/2, JNK and mTORC1 signaling pathways are involved in the insulin-mediated downregulation of human ABCA1 gene. Transcription factors LXRα, LXRß, FOXO1 and NF-κB are important contributors to this process, while FOXA2 does not regulate ABCA1 gene expression. Insulin causes the decrease in FOXO1, LXRα and LXRß binding to ABCA1 promoter, which is likely the cause of the decrease in the gene expression. Interestingly, the murine ABCA1 gene seems to be not regulated by insulin in hepatocytes (in vitro and in vivo). We suggest that the reason for this discrepancy is the difference in the 5'-regulatory regions of human and murine ABCA1 genes.

Adiponectin Stimulates Apolipoprotein A-1 Gene Expression in HepG2 Cells via AMPK, PPARα, and LXRs Signaling Mechanisms.

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and ß prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.

Inflammatory and autoimmune predictive markers of response to anti-PD-1/PD-L1 therapy in NSCLC and melanoma.

Immune checkpoint inhibitors (ICI) are a standard in cancer therapy, but few patients respond to the treatment. The aim of the present study was the determination of immunological markers for monitoring response to ICI. The present study included 74 patients receiving ICI in subsequent [group 1; non-small cell lung cancer (NSCLC)] and first-line setting (group 2; melanoma) and 30 patients with NSCLC receiving first-line chemotherapy. In groups 1 and 2 ß-2 microglobulin (B2-MG), neopterin (NPT), IL-6, IL-18, HLA-DRB1 and autoantibodies were assessed after two months of ICI, and before the start of next administration in group 3. In group 1 low level of B2-MG (P<0.0001), NPT (P<0.0001), IL-6 (P<0.0001), IL-18 (P=0.0003), HLA-DRB1*03 (P=0.016) and anti-TPO antibodies (P=0.016) were associated with response >six months. In group 2 high level of B2-MG (P=0.0001), NPT (P=0.0016), IL-6 (P=0.013) and IL-18 (P=0.032) were associated with early disease progression (

Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

Regulation of Apolipoprotein A-I Gene Expression in Human Macrophages by Oxidized Low-Density Lipoprotein.

Apolipoprotein A-I (ApoA-I) is a key component of reverse cholesterol transport in humans. In the previous studies, we demonstrated expression of the apoA-I gene in human monocytes and macrophages; however, little is known on the regulation of the apoA-I expression in macrophages during the uptake of modified low-density lipoprotein (LDL), which is one of the key processes in the early stages of atherogenesis leading to formation of foam cells. Here, we demonstrate a complex nature of the apoA-I regulation in human macrophages during the uptake of oxidized LDL (oxLDL). Incubation of macrophages with oxLDL induced expression of the apoA-I gene within the first 24 hours, but suppressed it after 48 h. Both effects depended on the interaction of oxLDL with the TLR4 receptor, rather than on the oxLDL uptake by the macrophages. The oxLDL-mediated downregulation of the apoA-I gene depended on the ERK1/2 and JNK cascades, as well as on the NF-κB cascade.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates.

BACKGROUND: The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI. AIM: To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs). METHODS: Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle. RESULTS: Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones. CONCLUSION: Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.

Efficacy of lorlatinib in lung carcinomas carrying distinct ALK translocation variants: The results of a single-center study.

BACKGROUND: Lorlatinib is a novel potent ALK inhibitor, with only a few studies reporting the results of its clinical use. METHODS: This study describes the outcomes of lorlatinib treatment for 35 non-small cell lung cancer patients with ALK rearrangements, who had 2 (n = 5), 1 (n = 26) or none (n = 4) prior tyrosine kinase inhibitors and received lorlatinib mainly within the compassionate use program. RESULTS: Objective tumor response (OR) and disease control (DC) were registered in 15/35 (43%) and 33/35 (94%) patients, respectively; brain metastases were particularly responsive to the treatment (OR: 22/27 (81%); DC: 27/27 (100%)). Median progression free survival (PFS) was estimated to be 21.8 months, and median overall survival (OS) approached to 70.1 months. Only 4 out of 35 patients experienced no adverse effects; two of them were the only subjects who had no clinical benefit from lorlatinib. PFS and OS in the no-adverse-events lorlatinib users were strikingly lower as compared to the remaining patients (1.1 months vs. 23.7 months and 10.5 months vs. not reached, respectively; p < 0.0001 for both comparisons). ALK translocation variants were known for 28 patients; there was no statistical difference between patients with V.1 and V.3 rearrangements with regard to the OS or PFS. CONCLUSION: Use of lorlatinib results in excellent disease outcomes, however caution must be taken for patients experiencing no adverse effects from this drug.

Comparative study of blood pressure and echocardiographic values of male cynomolgus macaques (Macaca fascicularis).

BACKGROUND: Primates represent a unique object for biomedical research, in particular in the field of physiology and pathology of the cardiovascular system. Echocardiography is the most important non-invasive method for the intravital study of the heart structure and function, intracardiac and systemic haemodynamics. The available data on reference values of echocardiographic parameters in primates are limited. METHODS: We determined and described 29 structural and functional parameters in echocardiographic examination using B-mode (two-dimensional scanning), M-mode (one-dimensional scanning) and in various Doppler modes together with blood pressure in 17 male cynomolgus macaques with an average age of 5.7 ± 0.6 years. We compared available literature data on reference values of echocardiography in this species. RESULTS AND CONCLUSIONS: Echocardiographic values in cynomolgus macaques depend on age, sex composition and the anaesthesia method. There is lack of presentation in the published studies of complete list of parameters that can be obtained by echocardiographic examination.

Rapid Improvement of the Performance Status and Reduction of the Tumor Size in KRAS-Mutated Colorectal Cancer Patient Receiving Binimetinib, Hydroxychloroquine, and Bevacizumab.

Activating RAS mutations occur in more than a half of colorectal cancers (CRCs). RAS-mutated CRCs are notoriously difficult to treat given that they are characterized by the aggressive disease course and the lack of appropriate targeted therapies. Recent preclinical studies demonstrated that RAS-mutated cells escape from therapeutic MEK inhibition by the development of autophagy, and this escape may be prevented by the administration of an antimalarial drug, hydroxychloroquine. The available clinical data are limited to a single case observation involving a patient with KRAS-mutated pancreatic cancer. Here, we report a woman with KRAS G12D-mutated CRC, whose tumor did not respond to conventional therapy. The combination of binimetinib, hydroxychloroquine, and bevacizumab was administered as a last-hope option. The patient experienced rapid improvement of the performance status. The tumor lumps demonstrated 17% reduction in the size within the first 6 weeks of the therapy. This report calls for evaluation of the efficacy of a combination of MEK inhibitors and hydroxychloroquine, possibly with the addition of bevacizumab, in chemotherapy-resistant patients with RAS-mutated cancers.

Capmatinib in MET Exon 14-Mutated or MET-Amplified Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), MET exon 14 skipping mutations occur in 3 to 4% and MET amplifications occur in 1 to 6%. Capmatinib, a selective inhibitor of the MET receptor, has shown activity in cancer models with various types of MET activation. METHODS: We conducted a multiple-cohort, phase 2 study evaluating capmatinib in patients with MET-dysregulated advanced NSCLC. Patients were assigned to cohorts on the basis of previous lines of therapy and MET status (MET exon 14 skipping mutation or MET amplification according to gene copy number in tumor tissue). Patients received capmatinib (400-mg tablet) twice daily. The primary end point was overall response (complete or partial response), and the key secondary end point was response duration; both end points were assessed by an independent review committee whose members were unaware of the cohort assignments. RESULTS: A total of 364 patients were assigned to the cohorts. Among patients with NSCLC with a MET exon 14 skipping mutation, overall response was observed in 41% (95% confidence interval [CI], 29 to 53) of 69 patients who had received one or two lines of therapy previously and in 68% (95% CI, 48 to 84) of 28 patients who had not received treatment previously; the median duration of response was 9.7 months (95% CI, 5.6 to 13.0) and 12.6 months (95% CI, 5.6 to could not be estimated), respectively. Limited efficacy was observed in previously treated patients with MET amplification who had a gene copy number of less than 10 (overall response in 7 to 12% of patients). Among patients with MET amplification and a gene copy number of 10 or higher, overall response was observed in 29% (95% CI, 19 to 41) of previously treated patients and in 40% (95% CI, 16 to 68) of those who had not received treatment previously. The most frequently reported adverse events were peripheral edema (in 51%) and nausea (in 45%); these events were mostly of grade 1 or 2. CONCLUSIONS: Capmatinib showed substantial antitumor activity in patients with advanced NSCLC with a MET exon 14 skipping mutation, particularly in those not treated previously. The efficacy in MET-amplified advanced NSCLC was higher in tumors with a high gene copy number than in those with a low gene copy number. Low-grade peripheral edema and nausea were the main toxic effects. (Funded by Novartis Pharmaceuticals; GEOMETRY mono-1 ClinicalTrials.gov number, NCT02414139.).

Treating non-small cell lung cancer with selumetinib: an up-to-date drug evaluation.

INTRODUCTION: RAS-RAF-MEK-ERK signaling is implicated in tumor development by promoting cell proliferation and other cancer hallmarks. MEK1/2 kinases are up-regulated in the majority of human cancers due to activation of tyrosine kinase receptors, RAS proteins, BRAF kinase, or some other members of the MAPK pathway. Targeting of MEK1/2 kinases may counterbalance cancer progression. AREAS COVERED: The authors analyze the scientific publications relevant to selumetinib (AZD6244, ARRY-142886) systematically and provide their expert opinion. EXPERT OPINION: Selumetinib is an oral selective allosteric inhibitor of MEK1 and MEK2 kinases. Single-agent selumetinib is usually administered in hydrogen sulfate capsules 75 mg twice a day; combination with other therapeutic compounds may or may not require reduced dosing of this drug. The established dose for pediatric patients is 25 mg per square meter twice a day. Selumetinib was extensively evaluated in non-small cell lung cancer (NSCLC) patients. Studies utilizing this drug as a monotherapy did not confirm its efficacy toward NSCLC. A phase II trial showed that the addition of selumetinib to docetaxel improved response rates and progression-free survival (PFS) in chemotherapy-pretreated KRAS-mutated NSCLC patients; however, a subsequent phase III study did not confirm these findings. There are several highly successful non-NSCLC selumetinib trials involving, e.g., patients with neurofibromatosis type 1 related tumors and children with low-grade BRAF-driven gliomas.

Exploratory analysis of front-line therapies in REVEL: a randomised phase 3 study of ramucirumab plus docetaxel versus docetaxel for the treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy.

INTRODUCTION: Non-small-cell lung cancer (NSCLC) is a heterogeneous disease. Front-line therapy may affect responses to subsequent treatment regimens, thus influencing second-line therapy decision making. In the randomised phase 3 REVEL study, second-line ramucirumab plus docetaxel (ram+doc) versus docetaxel (doc) improved survival of patients with metastatic NSCLC. We explore efficacy, safety and quality-of-life (QoL) in REVEL based on front-line therapy. METHODS: Patients were grouped by specific front-line therapy received. Overall survival (OS), progression-free survival (PFS), objective response rate, safety and QoL were assessed descriptively. Kaplan-Meier estimation and Cox proportional hazards modelling were used; frequencies reported in percentages. RESULTS: Baseline characteristics of 1253 patients were generally well balanced between treatment arms within each front-line therapy subgroup. For patients with non-squamous disease (n=912), induction therapies included platinum-based chemotherapy plus a taxane (n=227; 25%) or pemetrexed (n=449; 49%), with (n=172; 19%) or without bevacizumab. For patients with squamous disease (n=328), induction therapies included platinum-based chemotherapy plus gemcitabine (n=176; 54%) or a taxane (n=69; 21%). A highly selected subgroup (n=127; 14%) received pemetrexed continuation maintenance therapy. Ram+doc improved median OS and PFS versus doc across front-line therapy subgroups, as reflected by HRs ranging from 0.78 to 0.91 and 0.66 to 0.92, respectively, similar to results in the overall intention-to-treat cohort (HRs: 0.86 and 0.76, respectively). High-grade treatment-emergent adverse events of special interest (including neutropenia, febrile neutropenia, leucopenia and hypertension) were generally higher in ram+doc-treated patients relative to doc-treated patients regardless of front-line therapy. No clear differences in safety or QoL were seen across front-line therapy subgroups; outcomes were consistent with those reported in the overall intention-to-treat cohort. CONCLUSIONS: Results of this exploratory analysis suggest that second-line ram+doc may be effective regardless of prior treatment with platinum-based chemotherapy plus a taxane, pemetrexed, gemcitabine or bevacizumab. Overall, ram+doc is clinically beneficial across a wide range of patients with metastatic NSCLC who have progressed after various front-line therapies. TRIAL REGISTRATION NUMBER: NCT01168973.

EGFR T790M Mutation in TKI-Naïve Clinical Samples: Frequency, Tissue Mosaicism, Predictive Value and Awareness on Artifacts.

BACKGROUND: This study evaluated the distribution of epidermal growth factor receptor (EGFR) T790M mutations in treatment-naïve tumor and normal samples obtained from cancer patients. METHODS: We utilized allele-specific PCR (AS-PCR), digital droplet PCR (ddPCR) and next generation sequencing (NGS) to detect EGFR T790M allele in several collections of tumor and normal human tissues. RESULTS: AS-PCR analysis of treatment-naïve tumor samples revealed somatic T790M mutation in 3/394 (1%) non-small cell lung carcinomas (NSCLC) carrying the tyrosine kinase inhibitor (TKI)-sensitizing EGFR mutation, but in none of 334 NSCLC lacking EGFR exon 19 deletions (ex19del) or L858R substitutions and in none of 235 non-lung tumors. Use of highly sensitive and quantitative assays, such as ddPCR and NGS, produced a high number of T790M-specific signals even in presumably T790M-negative DNA specimens. This background noise was evidently higher in degraded DNA isolated from formalin-fixed paraffin-embedded tissues as compared to high molecular weight DNA. A combination of AS-PCR, ddPCR and NGS revealed mosaic EGFR T790M allele in 2/68 (3%) NSCLC treated with the first-generation TKI. Both these tumors produced evident and durable response to gefitinib. CONCLUSION: Detection of mosaic EGFR T790M mutation in treatment-naïve samples may be compromised by yet unresolved technical issues and may have limited clinical value.

Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants.

Multiple laboratory evidences indicate that distinct variants of ALK translocations differ in their biochemical properties and responsiveness to ALK tyrosine kinase inhibitors (TKIs). These data are supported by some clinical studies, which showed improved responses to crizotinib in non-small cell lung cancer (NSCLC) patients carrying particular variants of ALK translocation. We retrospectively considered 64 Russian patients with ALK-rearranged NSCLC, who were treated by crizotinib (n = 23), ceritinib (n = 39) or alectinib (n = 2). ALK fusion variants were genotyped by PCR. Median progression-free survival (PFS) approached to 18 and 21 months in subjects with "short" (v.3a/b, v.5a/b) vs. "long" (TAPE-domain containing) fusion variants (p = 0.783), respectively; similar data were obtained while comparing EML4/ALK variant 1 vs. other ALK translocations (19 and 21 months, respectively; p = 0.604). Objective response rates were also strikingly similar in the above groups ("short": 88%, "long": 77%, p = 0.479; variant 1: 76%, other translocations: 81%, p = 0.753). Furthermore, ALK variants did not influence the disease outcomes when patients treated by crizotinib and ceritinib were analyzed separately. Overall, PFS on ALK TKI did not depend on whether the drug was administered upfront or after chemotherapy. Ceritinib produced significantly longer PFS than crizotinib (p = 0.022). In conclusion, this study revealed that distinct ALK translocation variants render similar clinical responsiveness to ALK inhibitors.

Insulin downregulates C3 gene expression in human HepG2 cells through activation of PPARγ.

C3 is an acute phase protein, and thus its plasma concentration increases quickly and drastically during the onset of inflammation. Insulin plays a complex role in inflammation. Elevated level of plasma C3 was shown to correlate with heightened fasting insulin levels and insulin resistance and appears to be a risk factor for the cardiovascular disease and atherosclerosis. The main source of plasma C3 is liver. Nothing is known about effects of insulin on C3 gene expression and protein secretion by hepatocytes. In light of these data we asked if insulin is capable of regulating C3 production in hepatocytes. Here we show that insulin downregulates C3 gene expression in human hepatoma cells HepG2 through activation of PI3K, mTORC1, p38 and MEK1/2 signaling pathways. Transcription factors PPARα, PPARγ, HNF4α and NF-κB are important contributors to this process. Insulin activates PPARγ through PI3K/Akt/mTORC1 pathway, which results in PPARγ binding to DR4 and DR0 cis-acting elements within the C3 promoter and subsequent displacement of HNF4α and PPARα from these sites. As a result PPARα/NF-κB complex, which exists on C3 promoter, is broken down and C3 gene expression is downregulated. The data obtained can potentially be used to explain the molecular mechanism underlying the correlation between heightened level of plasma C3 and insulin resistance in humans.

Tumor necrosis factor α stimulates endogenous apolipoprotein A-I expression and secretion by human monocytes and macrophages: role of MAP-kinases, NF-κB, and nuclear receptors PPARα and LXRs.

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRß by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRß binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation.