Diagnostics of IDH1/2 Mutations in Intracranial Chondroid Tumors: Comparison of Molecular Genetic Methods and Immunohistochemistry.

Intracranial chondroid tumors are a heterogeneous group of neoplasms characterized by the presence of a cartilage matrix. These tumors exhibit overlapping clinical and histological features. Mutations in IDH1/2 genes serve as important diagnostic markers of tumor type, particularly chondrosarcoma. To improve the accuracy of IDH1/2 diagnostics, we compared three methods: biochip assay, real-time PCR with DNA melting analysis using TaqMan probes and sequencing (qPCR-DMA-Sanger), and immunohistochemistry (IHC). Tumor samples from 96 patients were investigated. The IDH1 mutations were detected in 34/64 (53%) chondrosarcomas; IHC detected 27/56 (48.2%) mutations, the qPCR-DMA-Sanger method 27/59 (46%) mutations, and the biochip assay revealed 29/60 (48.3%) mutations. The detection of IDH1 mutations in chordoma (2/15) and osteosarcoma (2/7) suggested the need for a revised diagnosis. In benign tumors, IDH1 mutations were present in chondroma (4/6), but absent in chondromyxoid fibroma (0/4). The most frequent IDH1 mutations were R132C (60%), R132L, and R132G (13.5% each), R132H (8%), and R132S (5%). The concordance between the biochip assay and IHC was 90%, between IHC and PCR-DMA-Sanger 83%, and between biochip assay and qPCR-DMA-Sanger was 98%, respectively. No IDH2 mutations were found. The use of independent diagnostic methods may improve the detection of IDH-mutant specimens in chondroid tumors.

The Influence of Structural Variants of the CES1 Gene on the Pharmacokinetics of Enalapril, Presumably Due to Linkage Disequilibrium with the Intronic rs2244613.

Variants in the CES1 gene encoding carboxylesterase 1 may affect the metabolism of enalapril to the active metabolite enalaprilat. It was shown that the A allele of rs71647871 and the C allele of rs2244613 led to a decrease in plasma enalaprilat concentrations. This study aimed to estimate the effect of structural haplotypes of CES1 containing the pseudogene CES1P1, or a hybrid of the gene and the pseudogene CES1A2, on the pharmacokinetics of enalapril. We included 286 Caucasian patients with arterial hypertension treated with enalapril. Genotyping was performed using real-time PCR and long-range PCR. Peak and trough plasma enalaprilat concentrations were lower in carriers of CES1A2. The studied haplotypes were in linkage disequilibrium with rs2244613: generally, the A allele was in the haplotype containing the CES1P1, and the C allele was in the haplotype with the CES1A2. Thus, carriers of CES1A2 have reduced CES1 activity against enalapril. Linkage disequilibrium of the haplotype containing the CES1P1 or CES1A2 with rs2244613 should be taken into account when genotyping the CES1 gene.

Transcriptome Analysis of Human Glioblastoma Cells Susceptible to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Glioblastoma multiforme (GBM) accounts for almost half of all primary malignant brain tumors in adults and has a poor prognosis. Here we demonstrated the oncolytic potential of the L-16 vaccine strain of measles virus (MV) against primary human GBM cells and characterized the genetic patterns that determine the sensitivity of primary human GBM cells to oncolytic therapy. MV replicated in all GBM cells, and seven out of eight cell lines underwent complete or partial oncolysis. RNA-Seq analysis identified about 1200 differentially expressed genes (FDR < 0.05) with at least two-fold expression level change between MV-infected and uninfected cells. Among them, the most significant upregulation was observed for interferon response, apoptosis and cytokine signaling. One out of eight GBM cell lines was defective in type I interferon production and, thus, in the post-interferon response, other cells lacked expression of different cellular defense factors. Thus, none of the cell lines displayed induction of the total gene set necessary for effective inhibition of MV replication. In the resistant cells, we detected aberrant expression of metalloproteinase genes, particularly MMP3. Thus, such genes could be considered intriguing candidates for further study of factors responsible for cell sensitivity and resistance to L-16 MV infection.

Genetic Association Study and Machine Learning to Investigate Differences in Platelet Reactivity in Patients with Acute Ischemic Stroke Treated with Aspirin.

Aspirin resistance (AR) is a pressing problem in current ischemic stroke care. Although the role of genetic variations is widely considered, the data still remain controversial. Our aim was to investigate the contribution of genetic features to laboratory AR measured through platelet aggregation with arachidonic acid (AA) and adenosine diphosphate (ADP) in ischemic stroke patients. A total of 461 patients were enrolled. Platelet aggregation was measured via light transmission aggregometry. Eighteen single-nucleotide polymorphisms (SNPs) in ITGB3, GPIBA, TBXA2R, ITGA2, PLA2G7, HMOX1, PTGS1, PTGS2, ADRA2A, ABCB1 and PEAR1 genes and the intergenic 9p21.3 region were determined using low-density biochips. We found an association of rs1330344 in the PTGS1 gene with AR and AA-induced platelet aggregation. Rs4311994 in ADRA2A gene also affected AA-induced aggregation, and rs4523 in the TBXA2R gene and rs12041331 in the PEAR1 gene influenced ADP-induced aggregation. Furthermore, the effect of rs1062535 in the ITGA2 gene on NIHSS dynamics during 10 days of treatment was found. The best machine learning (ML) model for AR based on clinical and genetic factors was characterized by AUC = 0.665 and F1-score = 0.628. In conclusion, the association study showed that PTGS1, ADRA2A, TBXA2R and PEAR1 polymorphisms may affect laboratory AR. However, the ML model demonstrated the predominant influence of clinical features.

The Influence of the CES1 Genotype on the Pharmacokinetics of Enalapril in Patients with Arterial Hypertension.

The angiotensin-converting enzyme inhibitor enalapril is hydrolysed to an active metabolite, enalaprilat, in the liver via carboxylesterase 1 (CES1). Previous studies show that variant rs71647871 in the CES1 gene affects the pharmacokinetics of enalapril on liver samples as well as healthy volunteers. This study included 286 Caucasian patients with arterial hypertension who received enalapril. The concentrations of enalapril and enalaprilat were determined before subsequent intake of the drug and 4 h after it with high-performance liquid chromatography (HPLC) and mass spectrometric detection. The study included genetic markers as follows: rs2244613, rs71647871 (c.428G>A, p.G143E) and three SNPs indicating the presence of a subtype CES1A1c (rs12149368, rs111604615 and rs201577108). Mean peak and trough enalaprilat concentrations, adjusted by clinical variables, were significantly lower in CES1 rs2244613 heterozygotes (by 16.6% and 19.6%) and in CC homozygotes (by 32.7% and 41.4%) vs. the AA genotype. In CES1A1c homozygotes, adjusted mean enalaprilat concentrations were 75% lower vs. heterozygotes and wild-type (WT) homozygotes. Pharmacogenetic markers of the CES1 gene may be a promising predictor for individualisation when prescribing enalapril.

The Susceptibility of Human Melanoma Cells to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.

Biochip-based approach for comprehensive pharmacogenetic testing.

OBJECTIVES: Individual sensitivity to many widely used drugs is significantly associated with genetic factors. The purpose of our work was to develop an instrument for simultaneous determination of the most clinically relevant pharmacogenetic markers to allow personalized treatment, mainly in patients with cardiovascular diseases. METHODS: Multiplex one-step polymerase chain reaction (PCR) followed by hybridization on a low-density biochip was applied to interrogate 15 polymorphisms in the following eight genes: VKORC1 -1639 G>A, CYP4F2 1297 G>A, GGCX 2374 C>G, CYP2C9 *2,*3 (430 C>T, 1075 A>C), CYP2D6 *3,*4, *6, *9, *41 (2549delA, 1846 G>A, 1707delT, 2615_2617delAAG, 2988 G>A), CYP2C19 *2,*3,*17 (681 G>A, 636 G>A, -806 C>T), ABCB1 (3435 C>T), SLCO1B1 *5. RESULTS: Two hundred nineteen patients with cardiovascular diseases (CVD) and 48 female patients with estrogen receptor (ER)-positive breast cancer (BC) were genotyped. Of the 219 CVD patients, 203 (92.7%) carried one or more actionable at-risk genotypes based on VKORC1/CYP2C9, CYP2C9, CYP2C19, SLCO1B1, and CYP2D6 genotypes. Among them, 67 patients (30.6%) carried one, 58 patients (26.5%) carried two, 51 patients (23.3%) carried three, 26 patients (11.9%) carried four, and one patient (0.4%) carried five risk actionable genotypes. In the ER-positive BC group 12 patients (25%) were CYP2D6 intermediate or poor metabolizers. CONCLUSIONS: The developed biochip is applicable for rapid and robust genotyping of patients who were taking a wide spectrum of medications to optimize drugs and dosage and avoid adverse drug reactions in cardiology, oncology, psychiatry, rheumatology and gastroenterology.

Reduced vs. standard dose native E. coli-asparaginase therapy in childhood acute lymphoblastic leukemia: long-term results of the randomized trial Moscow-Berlin 2002.

PURPOSE: Favorable outcomes were achieved for children with acute lymphoblastic leukemia (ALL) with the first Russian multicenter trial Moscow-Berlin (ALL-MB) 91. One major component of this regimen included a total of 18 doses of weekly intramuscular (IM) native Escherichia coli-derived asparaginase (E. coli-ASP) at 10000 U/m2 during three consolidation courses. ASP was initially available from Latvia, but had to be purchased from abroad at substantial costs after the collapse of Soviet Union. Therefore, the subsequent trial ALL-MB 2002 aimed at limiting costs to a reasonable extent and also at reducing toxicity by lowering the dose for standard risk (SR-) patients to 5000 U/m2 without jeopardizing efficacy. METHODS: Between April 2002 and November 2006, 774 SR patients were registered in 34 centers across Russia and Belarus, 688 of whom were randomized. In arm ASP-5000 (n = 334), patients received 5000 U/m2 and in arm ASP-10000 (n = 354) 10 000 U/m2 IM. RESULTS: Probabilities of disease-free survival, overall survival and cumulative incidence of relapse at 10 years were comparable: 79 ± 2%, 86 ± 2% and 17.4 ± 2.1% (ASP-5000) vs. 75 ± 2% and 82 ± 2%, and 17.9 ± 2.0% (ASP-10000), while death in complete remission was significantly lower in arm ASP-5000 (2.7% vs. 6.5%; p = 0.029). CONCLUSION: Our findings suggest that weekly 5000 U/m2E. coli-ASP IM during consolidation therapy are equally effective, more cost-efficient and less toxic than 10000 U/m2 for SR patients with childhood ALL.

Melanoma arising in a Giant congenital melanocytic nevus: two case reports.

BACKGROUND: A giant congenital melanocytic nevus (GCMN) is found in 0.1% of live-born infants. If present, the lesion has a chance of about 6% to develop into malignant melanoma. Both children and adults can be affected by malignant melanoma arising in a giant congenital nevus. Up to 95% of GCMNs harbor NRAS mutations, and mutations in the BRAF, MC1R, TP53, and GNAQ genes have also been described. The individualization of therapy is required, but diagnostic and prognostic criteria remain controversial. CASE PRESENTATIONS: We report two cases: 1) melanoma arising in a giant congenital nevus during the first month of life complicated with neurocutaneous melanosis (NCM), and 2) melanoma arising in a giant congenital nevus during the first 6 months of life. Pathology, immunohistochemistry, and genetic analyses of tumor tissue were performed. The first case revealed only a non-pathogenic P72R polymorphism of the TP53 gene in the homozygote condition. For the second case, a Q61K mutation was detected in the NRAS gene. CONCLUSION: Malignant melanoma associated with GCMN is rare and therefore poorly understood. Outcomes have been linked to the stage at diagnosis, but no additional pathological prognostic factors have been identified. The most frequent genetic event in giant CMNs is NRAS mutations, which was discovered in one of our cases. To accumulate evidence to improve disease prognosis and outcomes, children with congenital melanocytic nevus should be included in a systemic follow-up study from birth.

Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis.

Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

Autophagy inhibitors chloroquine and LY294002 enhance temozolomide cytotoxicity on cutaneous melanoma cell lines in vitro.

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

CrossHub: a tool for multi-way analysis of The Cancer Genome Atlas (TCGA) in the context of gene expression regulation mechanisms.

The contribution of different mechanisms to the regulation of gene expression varies for different tissues and tumors. Complementation of predicted mRNA-miRNA and gene-transcription factor (TF) relationships with the results of expression correlation analyses derived for specific tumor types outlines the interactions with functional impact in the current biomaterial. We developed CrossHub software, which enables two-way identification of most possible TF-gene interactions: on the basis of ENCODE ChIP-Seq binding evidence or Jaspar prediction and co-expression according to the data of The Cancer Genome Atlas (TCGA) project, the largest cancer omics resource. Similarly, CrossHub identifies mRNA-miRNA pairs with predicted or validated binding sites (TargetScan, mirSVR, PicTar, DIANA microT, miRTarBase) and strong negative expression correlations. We observed partial consistency between ChIP-Seq or miRNA target predictions and gene-TF/miRNA co-expression, demonstrating a link between these indicators. Additionally, CrossHub expression-methylation correlation analysis can be used to identify hypermethylated CpG sites or regions with the greatest potential impact on gene expression. Thus, CrossHub is capable of outlining molecular portraits of a specific gene and determining the three most common sources of expression regulation: promoter/enhancer methylation, miRNA interference and TF-mediated activation or repression. CrossHub generates formatted Excel workbooks with the detailed results. CrossHub is freely available athttps://sourceforge.net/projects/crosshub/.

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells.

BACKGROUND: The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the "Warburg effect" and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear. RESULTS: The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis. CONCLUSIONS: We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel biochips.

Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.

Control of thrombotic thrombocytopenic purpura by sirolimus in a child with juvenile myelomonocytic leukemia and somatic N-RAS mutation.

We describe an infant who developed juvenile myelomonocytic leukemia (JMML) at the age of 6 months. Myeloproliferation was effectively controlled by low-dose cytosine arabinoside and 13-cis retinoic acid therapy. Two years after therapy for JMML was stopped, at the age of 5 years, the patient developed autoimmune thrombotic thrombocytopenic purpura (TTP). TTP was transiently controlled by plasma exchange, prednisolone, rituximab, and cyclophosphamide, but relapsed within a short time. Long-term control of TTP was established by sirolimus. Somatic N-RAS G38A→Gly13Asp substitution was restricted to hematopoietic cells. The somatic N-RAS mutation may link myeloproliferation and autoimmunity.

Gel-based microarrays in clinical diagnostics in Russia.

Immobilization of molecular probes in 3D hydrogel elements provides some essential advantages compared with conventional flat surfaces. In this article, an integrated technology based on the use of low-density microarrays comprised of hemispherical gel elements, developed at the Engelhardt Institute of Molecular Biology (Moscow, Russia) for various applications will be reviewed. The structure of the gel can be adapted for immobilization of virtually any biological molecules in a natural hydrophilic environment. The discrimination between matching and mismatching duplexes of nucleic acids in these conditions is more reliable than on conventional flat surfaces, minimizing the number of elements needed to detect specific sequences. Protein molecules immobilized in hydrogel-based biochips better preserve their biological properties. As described in this article, such biochips were successfully applied for laboratory diagnostics in a wide variety of clinical conditions involving the identification of bacterial and viral pathogens, cancer-related mutations and protein tumor markers.

The role of DNA diffusion in solid phase polymerase chain reaction with gel-immobilized primers in planar and capillary microarray format.

The solid phase polymerase chain reaction (PCR) on a gel-based microarray system was studied under various durations of individual stages of the PCR cycle and spatial restriction of the reaction volume. Combining the experimental study with numerical modeling, we demonstrated that the diffusion of the PCR product in and out of a microarray element during the annealing and melting stages, respectively, is the main factor responsible for distinctive features of the studied type of PCR. The restriction of reaction volume leads to faster PCR signal growth. Particularly, the capillary array, whereby gel-based microarray elements are located on a glass bar inserted into capillary chamber, was found to be a suitable format for the development of the platform.