KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas.

This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the "hot" codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594-596: 38; codons 597-602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients' age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples.

The majority of NTRK1, NTRK2, and NTRK3 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 NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5'/3'-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5'/3'-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a normal 5'/3'-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1-3 analysis.

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

RET-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 RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

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. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5'/3'-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

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. RESULTS: Both the 5R allele of 30 bp VNTR and the 9R allele of 40 bp VNTR in SLC6A3 were associated with a reduced risk to tobacco smoking [odds ratio (OR) 0.53, 95% confidence interval (CI) 0.37-0.75; OR 0.62, 95% CI 0.43-0.88]. Although the carriers of 9R allele also had high Fagerström test for nicotine dependence scores (OR 1.65, 95% CI 1.04-2.60), they were still more likely to succeed in smoking cessation (OR 0.59, 95% CI 0.40-0.88). Also, current smokers had more than 2.5-fold likelihood to have increased SLC6A3 methylation levels than former smokers (OR 2.72, 95% CI 1.63-4.53). CONCLUSION: The SLC6A3 5R of 30 bp and 9R of 40 bp VNTR variants may lead to a reduced risk to start smoking through decreased dopamine availability, and can also affect the success in subsequent smoking cessation attempts. Moreover, the elevated mean methylation values in the first intron of SLC6A3 may be related to nicotine dependence via a more active dopamine transporter.

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, 0.6-15.8 years). RNA and cDNA were successfully obtained in 29 cases. The molecular analysis included sequential tests for 5'/3'-end unbalanced gene expression, variant-specific PCR, and next-generation sequencing (NGS). RESULTS: 5'/3'-end unbalanced ALK expression was revealed in 15/29 (52%) IMTs. Strikingly, all these tumors demonstrated high amount of ALK protein detected by immunohistochemistry. Variant-specific PCR was capable of identifying the type of ALK rearrangement in 11/15 IMTs with 5'/3'-end unbalanced ALK expression. The remaining four tumors were analyzed by NGS; two known and two novel (CLTC-ins6del84-ALK and EEF1G-ALK) ALK rearrangements were detected. Five IMTs demonstrated 5'/3'-end unbalanced ROS1 expression, and all these tumors carried TFG-ROS1 fusion. Nine tumors, which were negative for 5'/3'-end unbalanced ALK/ROS1 expression, were subjected to further analysis. Variant-specific PCR revealed two additional tumors with gene rearrangements (TFG-ROS1 and ETV6-NTRK3). The remaining seven IMTs were tested by NGS; single instances of TFG-ROS1 and novel SRF-PDGFRb translocations were detected. CONCLUSIONS: Twenty-four of 29 IMTs (83%) were shown to have druggable rearrangements involving tyrosine kinases, 20 of these 24 gene fusions were detectable by simple and inexpensive PCR assay, which is based on the detection 5'/3'-end unbalanced gene expression.

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. MATERIALS AND METHODS: We studied the 1460 C/T variation and the variable number tandem repeat polymorphism in the MAOA gene and A/G variation in intron 13 in the MAOB gene together with four DNA methylation sites in both of these genes in relation to several smoking-related phenotypes in a study population of 1230 Whites of Russian origin. RESULTS: The genotypes studied were found to be associated with smoking status in women; the MAOB G variant allele was more prevalent in female smokers than nonsmokers [odds ratio (OR): 2.16, 95% confidence interval (CI): 1.08-4.33], whereas a reverse relation was observed for the MAOA 1460 T-variant allele (OR: 0.44, 95% CI: 0.21-0.91) and variable number tandem repeat low-activity alleles (OR: 0.49, 95% CI: 0.24-0.98). Moreover, the mean methylation values of the CpG sites studied in the MAOA gene were related to smoking behavior in women. Similarly, several methylation patterns in the MAOB gene were associated with a smoking history, with each CpG site showing a remarkable sex dependence. CONCLUSION: Smoking behavior seems to be related to the genetic and epigenetic profile of MAO genes, with considerable individual and sex-related differences.

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. MATERIALS AND METHODS: We studied eight functional gene variants of one of the most important drug-metabolizing enzymes, CYP2D6, in relation to smoking behaviour in our well-characterized study population consisting of 1230 Whites of Russian origin. In addition, potential associations between methylation levels in a CpG island in the CYP2D6 gene and sex, age, different smoking-related phenotypes and CYP2D6 genotypes were studied. RESULTS: Both age and sex were found to be associated with the methylation level of the CYP2D6 gene. The CYP2D6 methylation pattern also showed high genotype dependence; compared with the extensive metabolizer genotype, the poor metabolizer genotype occurred notably more frequently with higher methylation status (odds ratio 5.05, 95% confidence interval 2.14-11.90). Moreover, higher methylation levels were found to be related inversely to heavier smoking (odds ratio 0.56, 95% confidence interval 0.35-0.91). We also found associations between the CYP2D6 genotype and smoking habits; the poor metabolizer genotype tended to decrease the risk of becoming a heavy smoker compared with the extensive metabolizers, whereas the ultrarapid metabolism-related genotypes tended to increase the risk. CONCLUSION: The CYP2D6-related metabolic capacity seems to be related to cigarette consumption both through genetic and through epigenetic mechanisms.

High prevalence of GPRC5A germline mutations in BRCA1-mutant breast cancer patients.

In a search for new breast cancer (BC) predisposing genes, we performed a whole exome sequencing analysis using six patient samples of familial BC and identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor GPRC5A. An extended case-control study revealed a tenfold enrichment for this mutation in BC patients carrying the 5382insC allele of BRCA1, the major founder mutation in the Russian population, compared to wild-type BRCA1 BC cases [6/117 (5.1%) vs. 8/1578 (0.5%), p = 0.0002]. In mammary tumors (n = 60), the mRNA expression of GPRC5A significantly correlated with that of BRCA1 (p = 0.00018). In addition, the amount of GPRC5A transcript was significantly lower in BC obtained from BRCA1 mutation carriers (n = 17) compared to noncarriers (n = 93) (p = 0.026). Accordingly, a siRNA-mediated knockdown of either BRCA1 or GPRC5A in the MDA-MB-231 human BC cell line reduced expression of GPRC5A or BRCA1, respectively. Knockdown of GPRC5A also attenuated radiation-induced BRCA1- and RAD51-containing nuclear DNA repair foci. Taken together, these data suggest that GPRC5A is a modifier of BC risk in BRCA1 mutation carriers and reveals a functional interaction of these genes.

Double heterozygotes among breast cancer patients analyzed for BRCA1, CHEK2, ATM, NBN/NBS1, and BLM germ-line mutations.

17 double heterozygous (DH) breast cancer (BC) patients were identified upon the analysis of 5,391 affected women for recurrent Slavic mutations in BRCA1, CHEK2, NBN/NBS1, ATM, and BLM genes. Double heterozygosity was found for BRCA1 and BLM (4 patients), BRCA1 and CHEK2 (4 patients), CHEK2 and NBS1 (3 patients), BRCA1 and ATM (2 patients), CHEK2 and BLM (2 patients), CHEK2 and ATM (1 patient), and NBS1 and BLM (1 patient). DH BC patients were on average not younger than single mutation carriers and did not have an excess of bilateral BC; an additional non-breast tumor was documented in two BRCA1/BLM DH patients (ovarian cancer and lymphoplasmacytic lymphoma). Loss-of-heterozygosity (LOH) analysis of involved genes was performed in 5 tumors, and revealed a single instance of somatic loss of the wild-type allele (LOH at CHEK2 locus in BRCA1/CHEK2 double heterozygote). Distribution of mutations in patients and controls favors the hypothesis on multiplicative interaction between at least some of the analyzed genes. Other studies on double heterozygosity for BC-predisposing germ-line mutations are reviewed.

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). AREAS COVERED: This review describes the spectrum of BRAF mutations and their functional roles, discusses treatment options available for BRAF p.V600 and non-V600 mutated NSCLCs, and identifies some gaps in the current knowledge. EXPERT OPINION: Administration of combined BRAF/MEK inhibitors usually produces significant, although often a short-term, benefit to NSCLC patients with BRAF V600 (class 1) mutations. There are no established treatments for BRAF class 2 (L597, K601, G464, G469A/V/R/S, fusions, etc.) and class 3 (D594, G596, G466, etc.) mutants, which account for up to two-thirds of BRAF-driven NSCLCs. Many important issues related to the use of immune therapy for the management of BRAF-mutated NSCLC deserve further investigation. The rare occurrence of BRAF mutations in NSCLC is compensated by high overall incidence of lung cancer disease; therefore, clinical studies on BRAF-associated NSCLC are feasible.

Response to trametinib, hydroxychloroquine, and bevacizumab in a young woman with NRAS-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 NRAS-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. The duration of this response was 4 months, but the patient died 10 months after the initiation of this triple therapy. This case report and the analysis of other available studies warrant further investigations on combined MEK and autophagy inhibition in RAS-mutated tumors.

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 FGFR1-4 gene rearrangements, detection of activating mutations located within FGFR1-4, IDH1/2, ERBB2 (HER2), KRAS, NRAS, BRAF, and PIK3CA genes, and measurement of the expression of ERBB2, PD-L1, and FGFR1-4 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). The NGS failure rate was 3/168 (1.8%). iCCAs, but not other categories of BTCs, were characterized by frequent FGFR2 alterations (17/82, 20.7%) and IDH1/2 mutations (23/82, 28%). Other potentially druggable events included ERBB2 amplifications or mutations (7/165, 4.2% of all successfully analyzed BTCs) and BRAF p.V600E mutations (3/165, 1.8%). In addition to NGS, we analyzed microsatellite instability (MSI) using the standard five markers and revealed this event in 3/158 (1.9%) BTCs. There were no instances of ALK, ROS1, RET, and NTRK1-3 gene rearrangements or MET exon 14 skipping mutations. Parallel analysis of 47 iCCA samples with the Illumina TruSight Tumor 170 kit confirmed good performance of our NGS panel. In conclusion, targeted RNA sequencing coupled with the 3' RACE technology is an efficient tool for the molecular diagnostics of BTCs.

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. According to the VGG19CRC model, adipose tissue scores were calculated for TCGA-CRC hematoxylin and eosin (H&E) images and images from patients at Zhujiang Hospital of Southern Medical University and First People's Hospital of Chenzhou. Kaplan-Meier (KM) analysis was used to compare the overall survival (OS) of patients. The XCell and MCP-Counter algorithms were used to evaluate the immune cell scores of the patients. Gene set enrichment analysis (GSEA) and single-sample GSEA (ssGSEA) were used to analyze upregulated and downregulated pathways. In TCGA-CRC, patients with high-adipocytes (high-ADI) CRC had significantly shorter OS times than those with low-ADI CRC. In a validation queue from Zhujiang Hospital of Southern Medical University (Local-CRC1), patients with high-ADI had worse OS than CRC patients with low-ADI. In another validation queue from First People's Hospital of Chenzhou (Local-CRC2), patients with low-ADI CRC had significantly longer OS than patients with high-ADI CRC. We developed a deep convolution network to segment various tissues from pathological H&E images of CRC and automatically quantify ADI. This allowed us to further analyze and predict the survival of CRC patients according to information from their segmented pathological tissue images, such as tissue components and the tumor microenvironment.

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. RESULTS: Variant-specific PCR tests for 4 common ALK and 15 common ROS1 translocations detected 115 (5.7%) and 44 (2.2%) rearrangements, respectively. Virtually all samples with common ALK fusions demonstrated some level of 5'/3' mRNA ends unbalanced expression, and 8 additional NSCLCs with rare ALK fusions were further identified by PCR or NGS among 48 cases selected based on ALK expression measurements. Interestingly, NSCLCs with unbalanced 5'-/3'-end ALK expression but without identified ALK translocations had elevated frequency of RAS mutations (21/40, 53%) suggesting the role of RAS activation in the alternative splicing of ALK gene. In contrast to ALK, only a minority of ROS1 translocation-positive cases demonstrated unbalanced gene expression, with both 5'- and 3'-end mRNA expression being elevated in most of the samples with translocations. Surprisingly, high ROS1 expression level was also found to be characteristic for NSCLCs with activating mutations in other tyrosine kinases such as EGFR, ALK, or MET. CONCLUSIONS: Comprehensive ALK analysis can be performed by the test for 5'-/3'-end unbalanced expression with minimal risk of missing an ALK rearrangement. In contrast, the use of the test for 5'-/3'-end unbalanced expression for the detection of ROS1 fusions is complicated; hence, the utilization of variant-specific PCR assays for ROS1 testing is preferable.

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 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). MET exon 14Δ was significantly associated with elderly age and non-smoking status of the patients. A total of 34 (97%) out of 35 tumors carrying MET exon 14Δ showed preferential expression of the mutated allele; this imbalance was attributed to the down-regulation of the expression of the wild-type gene copy. Sanger sequencing confirmed the presence of genomic exon 14 splice site mutations in 24/35 (68.6%) cases, which showed MET exon 14 skipping by PCR. In addition to LCs described above, some carcinomas demonstrated low-abundance MET exon 14Δ-specific signal. Low-level expression of MET exon 14Δ allele may potentially compromise the results of allele-specific PCR-based tests, therefore comparison of the level of expression of mutated and normal alleles is essential for the reliability of MET gene testing.

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.

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. METHODS: Nineteen patients were prospectively included in the study. RESULTS: Two (11%) patients experienced partial response (PR) and 11 (58%) subjects showed stable disease (SD). Median time to progression approached 6.1 months (range 1.6-15.0 months). Cetuximab efficacy did not correlate with RNA expression of EGFR and insulin-like growth factor 2 (IGF2). Only one tumor carried PIK3CA mutation, and this CRC responded to cetuximab. Exome analysis of patients with progressive disease (PD) revealed 1 CRC with high-level microsatellite instability and 1 instance of HER2 oncogene amplification; 3 of 4 remaining patients with PD had allergic reactions to cetuximab, while none of the subjects with PR or SD had this complication. Comparison with 19 retrospective KRAS/NRAS/BRAF mutation-negative patients receiving first-line fluoropyrimidines revealed no advantages or disadvantages of cetuximab therapy. CONCLUSIONS: Cetuximab demonstrates only modest efficacy when given as a first-line monotherapy to KRAS/NRAS/BRAF mutation-negative CRC patients. It is of question, why meticulous patient selection, which was undertaken in the current study, did not result in the improvement of outcomes of single-agent cetuximab treatment.

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.