Use of 3' Rapid Amplification of cDNA Ends (3' RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic Alterations in Urothelial Carcinomas.

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3' Rapid Amplification of cDNA Ends (3' RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, , , , , (), () and . -activating point mutations or fusions were found in 54/233 (23.

Response to trametinib, hydroxychloroquine, and bevacizumab in a young woman with -mutated metastatic intrahepatic cholangiocarcinoma: a case report.

Systemic chemotherapy is the main treatment option for patients with advanced intrahepatic cholangiocarcinoma (iCCA), however, its efficacy is limited. Herein, we report a young patient with -mutated chemoresistant metastatic iCCA, who received second-line therapy with a combination of trametinib (MEK1/2 inhibitor), hydroxychloroquine (autophagy inhibitor), and bevacizumab (angiogenesis inhibitor). A significant response was achieved during therapy, resulting in a 25% decrease in the size of tumor lesions after 2 months of treatment and an improvement in the patient's condition.

Pathways and targeting avenues of BRAF in non-small cell lung cancer.

Introduction: BRAF is a serine-threonine kinase implicated in the regulation of MAPK signaling cascade. BRAF mutation-driven activation occurs in approximately 2-4% of treatment-naive non-small cell carcinomas (NSCLCs). BRAF upregulation is also often observed in tumors with acquired resistance to receptor tyrosine kinase inhibitors (TKIs).

Molecular Analysis of Biliary Tract Cancers with the Custom 3' RACE-Based NGS Panel.

The technique 3' rapid amplification of cDNA ends (3' RACE) allows for detection of translocations with unknown gene partners located at the 3' end of the chimeric transcript. We composed a 3' RACE-based RNA sequencing panel for the analysis of gene rearrangements, detection of activating mutations located within , and genes, and measurement of the expression of and transcripts. This NGS panel was utilized for the molecular profiling of 168 biliary tract carcinomas (BTCs), including 83 intrahepatic cholangiocarcinomas (iCCAs), 44 extrahepatic cholangiocarcinomas (eCCAs), and 41 gallbladder adenocarcinomas (GBAs).

Cost-Efficient Detection of Gene Fusions: Single-Center Analysis of 8075 Tumor Samples.

The majority of , , and rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5'/3'-end expression imbalances is potentially capable of detecting the entire spectrum of gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis.

Rapid and Cost-Efficient Detection of Rearrangements in a Large Consecutive Series of Lung Carcinomas.

-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the gene; this test relies on the fact that translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance.

Deep Learning Analysis of the Adipose Tissue and the Prediction of Prognosis in Colorectal Cancer.

Research has shown that the lipid microenvironment surrounding colorectal cancer (CRC) is closely associated with the occurrence, development, and metastasis of CRC. According to pathological images from the National Center for Tumor diseases (NCT), the University Medical Center Mannheim (UMM) database and the ImageNet data set, a model called VGG19 was pre-trained. A deep convolutional neural network (CNN), VGG19CRC, was trained by the migration learning method.

Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process.

Comprehensive evaluation of the test for 5'-/3'-end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer.

Background: Despite the progress in the development of next-generation sequencing (NGS), diagnostic PCR assays remain to be utilized in clinical routine due to their simplicity and low cost. Tests for 5'-/3'-end mRNA unbalanced expression can be used for variant-independent detection of translocations, however, many technical aspects of this methodology require additional investigations.

Methods: Known ALK/ROS1 fusions and 5'-/3'-end unbalanced expression were analyzed in 2009 EGFR mutation-negative non-small cell lung cancer (NSCLC) samples with RT-PCR tests, which were optimized for the use with FFPE-derived RNA.

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%)).

The effect of SLC6A3 variable number of tandem repeats and methylation levels on individual susceptibility to start tobacco smoking and on the ability of smokers to quit smoking.

Objective: Nicotine acts through the dopamine pathway in the brain affecting reward processing through cigarette consumption. Thus, both genetic and epigenetic factors related to dopamine metabolism may influence individual's smoking behavior.

Materials And Methods: We studied variations of two variable numbers of tandem repeats (VNTRs), 40 and 30 bp in length, in SLC6A3 gene together with six DNA methylation sites located in a first intron of the gene in relation to several smoking-related phenotypes in a study population consisting of 1230 Whites of Russian origin.

Gene rearrangements in consecutive series of pediatric inflammatory myofibroblastic tumors.

Background: Inflammatory myofibroblastic tumors (IMTs) are exceptionally rare neoplasms, which are often driven by rearranged tyrosine kinases.

Methods: This study considered 33 consecutive patients with IMT (median age, 6.6; age range, 0.

Comparative analysis of expression of mutant and wild-type alleles is essential for reliable PCR-based detection of MET exon 14 skipping.

MET exon 14 skipping (exon 14Δ) mutations are associated with tumor sensitivity to a number of tyrosine kinase inhibitors, however clinical testing for MET gene status remains complicated. We developed a simple allele-specific PCR cDNA-based test, which allowed for the identification of MET exon 14Δ allele in 35 (2.5%) out of 1415 EGFR mutation-negative lung carcinomas (LCs).

PCR-based detection of EGFR, ALK, KRAS and BRAF mutations in Russian patients with lung adenocarcinoma: a single-center experience.

In contrast to other countries with predominantly white populations, Russian smoking-related lung cancers (LC) are mainly squamous cell carcinomas and approximately half lung adenocarcinomas (AdCa) are not related to tobacco consumption. Given that smoking significantly influences the probability of presence of actionable mutations in LC, one would expect that Russian lung AdCa patients would differ from other white populations in distribution of EGFR, ALK, KRAS and BRAF mutations. Herein, 2,336 consecutive lung AdCa cases, including 1,203 patients with known smoking status, were subjected to sequential testing for the above mutations.

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.

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).

First-Line Cetuximab Monotherapy in KRAS/NRAS/BRAF Mutation-Negative Colorectal Cancer Patients.

Background: Colorectal carcinomas (CRCs) are sensitive to treatment by anti-epidermal growth factor receptor (EGFR) antibodies only if they do not carry activating mutations in down-stream EGFR targets (KRAS/NRAS/BRAF). Most clinical trials for chemo-naive CRC patients involved combination of targeted agents and chemotherapy, while single-agent cetuximab or panitumumab studies included either heavily pretreated patients or subjects who were not selected on the basis of molecular tests. We hypothesized that anti-EGFR therapy would have significant efficacy in chemo-naive patients with KRAS/NRAS/BRAF mutation-negative CRC.

Spectrum of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies.

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.

The genotypes and methylation of MAO genes as factors behind smoking behavior.

Objective: Smoking dependence is the main cause for tobacco-related illnesses. The addiction-causing substance in tobacco, nicotine, acts through the dopamine pathway in the brain, causing several pleasurable experiences through cigarette smoking. Thus, both genetic and epigenetic factors related to dopamine metabolism may play an important role in influencing an individual's smoking behavior.

Survival Outcomes in EGFR Mutation-Positive Lung Cancer Patients Treated with Gefitinib until or beyond Progression.

Background: Discontinuation of gefitinib treatment is often accompanied by a disease flare. Some studies have demonstrated a benefit of the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) beyond progression; however, long-term results of these investigations remain limited.

Patients And Methods: We observed 70 patients with EGFR-mutated (EGFR-M+) non-small cell lung cancer (NSCLC) receiving single-agent gefitinib in a routine clinical setting; 56 patients were experiencing RECIST progression at the time of the analysis.

Distribution of EGFR Mutations in 10,607 Russian Patients with Lung Cancer.

Introduction: This study was aimed to evaluate distribution of epidermal growth factor receptor (EGFR) mutations in a large series of Russian lung cancer (LC) patients.

Methods: 10,607 LC samples were considered for EGFR analysis; EGFR status was successfully determined in 10,426 cases (98.3 %), indicating relatively low failure rate.

Effect of genotype and methylation of CYP2D6 on smoking behaviour.

Objective: Cigarette smoking is one of the most influential environmental factors affecting the DNA methylation patterns. The addiction-causing substance of tobacco smoke, nicotine, has also shown the potential to alter DNA methylation patterns. However, genetics has a strong influence on DNA methylation patterns, which in turn may affect an individual's smoking behaviour.