Geographic variation of mutagenic exposures in kidney cancer genomes.

International differences in the incidence of many cancer types indicate the existence of carcinogen exposures that have not yet been identified by conventional epidemiology make a substantial contribution to cancer burden1. In clear cell renal cell carcinoma, obesity, hypertension and tobacco smoking are risk factors, but they do not explain the geographical variation in its incidence2. Underlying causes can be inferred by sequencing the genomes of cancers from populations with different incidence rates and detecting differences in patterns of somatic mutations. Here we sequenced 962 clear cell renal cell carcinomas from 11 countries with varying incidence. The somatic mutation profiles differed between countries. In Romania, Serbia and Thailand, mutational signatures characteristic of aristolochic acid compounds were present in most cases, but these were rare elsewhere. In Japan, a mutational signature of unknown cause was found in more than 70% of cases but in less than 2% elsewhere. A further mutational signature of unknown cause was ubiquitous but exhibited higher mutation loads in countries with higher incidence rates of kidney cancer. Known signatures of tobacco smoking correlated with tobacco consumption, but no signature was associated with obesity or hypertension, suggesting that non-mutagenic mechanisms of action underlie these risk factors. The results of this study indicate the existence of multiple, geographically variable, mutagenic exposures that potentially affect tens of millions of people and illustrate the opportunities for new insights into cancer causation through large-scale global cancer genomics.

Impact of mechanical bowel preparation on the gut microbiome of patients undergoing left-sided colorectal cancer surgery: randomized clinical trial.

BACKGROUND: Postoperative complications after colorectal cancer surgery have been linked to the gut microbiome. However, the impact of mechanical bowel preparation using oral preparation agents or rectal enema on postoperative infections remains poorly understood. This study aimed to compare the impact of oral preparation and rectal enema on the gut microbiome and postoperative complications. METHODS: This open-label pilot RCT was conducted at the National Cancer Institute, Vilnius, Lithuania. Patients with left-side colorectal cancer scheduled for elective resection with primary anastomosis were randomized 1 : 1 to preoperative mechanical bowel preparation with either oral preparation or rectal enema. Stool samples were collected before surgery, and on postoperative day 6 and 30 for 16S rRNA gene sequencing analysis. The primary outcome was difference in ß-diversity between groups on postoperative day 6. RESULTS: Forty participants were randomized to oral preparation (20) or rectal enema (20). The two groups had similar changes in microbiome composition, and there was no difference in ß-diversity on postoperative day 6. Postoperative infections occurred in 12 patients (32%), without differences between the study groups. Patients with infections had an increased abundance of bacteria from the Actinomycetaceae family, Actinomyces genus, Sutterella uncultured species, and Enterococcus faecalis species. CONCLUSION: Mechanical bowel preparation with oral preparation or rectal enema resulted in similar dysbiosis. Patients who experienced postoperative infections exhibited distinct gut microbiome compositions on postoperative day 6, characterized by an increased abundance of bacteria from the Actinomycetaceae family, Actinomyces genus, Sutterella uncultured species, and Enterococcus faecalis species. REGISTRATION NUMBER: NCT04013841 (http://www.clinicaltrials.gov).

Combination of DNA methylation biomarkers with multiparametric magnetic resonance and ultrasound imaging fusion biopsy to detect the local spread of prostate cancer.

BACKGROUND: This study aimed to investigate the extent of field cancerization adjacent to index lesions in prostate cancer (PCa) by measuring DNA methylation of selected tumor suppressor genes in the perifocal tissue of PCa not visible on multiparametric magnetic resonanse imaging (mpMRI) for the safe zone of focal therapy identification. METHODS: A total of 272 patients were enrolled in this study, 44 patients' tissue biosamples were included in the field cancerization research, and 272 urine samples were included in the urine-based test development. Targeted biopsies were performed using the mpMRI/ultrasoundimage fusion system. RESULTS: Quantitative analysis revealed significantly higher DNA methylation levels of RARB, RASSF1, GSTP1 & APC genes in the index lesion compared with perifocal tissue samples 10 mm away from it (p < 0.0001). Notably, the RARB, GSTP1 & APC and RARB, RASSF1, GSTP1 & APC biomarker combinations exhibited the highest sensitivity and specificity comparing the extent of DNA methylation in index lesions and noncancerous prostate tissues 20 mm away (both area under the curve [AUC] = 0.98; p < 0.0001). The analysis of the potential urinary biomarkers showed that the combination of all four DNA methylation biomarkers with prostate-specific antigen (PSA) or PSA density (PSAD) in the blood significantly improves the detection of clinically significant PCa (csPCa). The combination of the four-biomarker test with PSAD allowed the identification of csPCa with ≥90% sensitivity and specificity. CONCLUSION: Thus, this study suggests that for focal therapy by region target hemi-ablation, the safe distance from the index lesion is no less than 10 mm. Noninvasive urine DNA methylation tests in combination with PSAD could be used for further follow-up of the patients, but larger prospective studies with external validation are needed.

Surveillance of cfDNA Hot Spot Mutations in NSCLC Patients during Disease Progression.

Non-small cell cancer (NSCLC) has been identified with a great variation of mutations that can be surveyed during disease progression. The aim of the study was to identify and monitor lung cancer-specific mutations incidence in cell-free DNA as well as overall plasma cell-free DNA load by means of targeted next-generation sequencing. Sequencing libraries were prepared from cell-free DNA (cfDNA) isolated from 72 plasma samples of 41 patients using the Oncomine Lung cfDNA panel covering hot spot regions of 11 genes. Sequencing was performed with the Ion Torrent™ Ion S5™ system. Four genes were detected with highest mutation incidence: KRAS (43.9% of all cases), followed by ALK (36.6%), TP53 (31.7%), and PIK3CA (29.3%). Seven patients had co-occurring KRAS + TP53 (6/41, 14.6%) or KRAS + PIK3CA (7/41, 17.1%) mutations. Moreover, the mutational status of TP53 as well an overall cell-free DNA load were confirmed to be predictors of poor progression-free survival (HR = 2.5 [0.8-7.7]; p = 0.029 and HR = 2.3 [0.9-5.5]; p = 0.029, respectively) in NSCLC patients. In addition, TP53 mutation status significantly predicts shorter overall survival (HR = 3.4 [1.2-9.7]; p < 0.001). We demonstrated that TP53 mutation incidence as well as a cell-free DNA load can be used as biomarkers for NSCLC monitoring and can help to detect the disease progression prior to radiological confirmation of the status.

Gingival Tissue MiRNA Expression Profiling and an Analysis of Periodontitis-Specific Circulating MiRNAs.

This study aimed to identify the microRNAs (miRNAs) associated with periodontitis (PD) in gingival tissues, and to evaluate the levels of these selected miRNAs in the saliva and blood plasma among participants with and without rheumatoid arthritis (RA). A genome-wide miRNA expression analysis in 16 gingival tissue samples revealed 177 deregulated miRNAs. The validation of the miRNA profiling results in 80 gingival tissue samples revealed that the PD-affected tissues had a higher expression of miR-140-3p and -145-5p, while the levels of miR-125a-3p were significantly lower in inflamed tissues. After a thorough validation, four miRNAs, namely miR-140-3p, -145-5p, -146a-5p, and -195-5p, were selected for further analysis in a larger sample of salivary (N = 173) and blood plasma (N = 221) specimens. Increased salivary levels of miR-145-5p were associated with higher mean values of pocket probing depth and bleeding on probing index. The plasma-derived levels of miR-140-3p were higher among the participants with PD. In conclusion, the gingival levels of miR-140-3p, -145-5p, and -125a-3p were independently associated with PD presence and severity. The salivary and blood plasma levels of the target miRNAs were diversely related to PD. Similar miRNA associations with PD were observed among the participants with and without RA.

ARID1A, NOTCH and WNT Signature in Gynaecological Tumours.

Ovarian cancer (OC) is among the deadliest gynaecologic malignancies in the world. The majority of OC patients are diagnosed at an advanced stage, with high-grade serous OC (HGSOC). The lack of specific symptoms and suitable screening strategies lead to short progression-free survival times in HGSOC patients. The chromatin-remodelling, WNT and NOTCH pathways are some of the most dysregulated in OC; thus their gene mutations and expression profile could serve as diagnostic or prognostic OC biomarkers. Our pilot study investigated mRNA expression of the SWI/SNF chromatin-remodelling complex gene ARID1A, NOTCH receptors, WNT pathway genes CTNNB1 and FBXW7 mRNA expression in two OC cell cultures as well as 51 gynaecologic tumour tissues. A four-gene panel consisting of ARID1A, CTNNB1, FBXW7 and PPP2R1A was used to investigate mutations in gynaecologic tumour tissue. All seven analysed genes were found to be significantly downregulated in OC when compared with non-malignant gynaecologic tumour tissues. NOTCH3 was also downregulated in SKOV3 cells when compared to A2780. Fifteen mutations were found in 25.5% (13/51) of the tissue samples. ARID1A predicted mutations were the most prevalent with alterations detected in 19% (6/32) HGSOC and 67% (6/9) of other OC cases. Thus, ARID1A and NOTCH/WNT-pathway-related changes could be useful diagnostic biomarkers in OC.

Germline DNA Damage Response Gene Mutations in Localized Prostate Cancer.

Background and Objectives: Germline DNA damage response (DDR) gene mutations correlate with increased prostate cancer (PCa) risk and a more aggressive form of the disease. DDR mutation testing is recommended for metastatic PCa cases, while eligible information about the mutations' burden in the early-stage localized PCa is still limited. This study is aimed at the prospective detection of DDR pathway mutations in cases with localized PCa and correlation with clinical, histopathological, and radiological data. A comparison to the previously assessed cohort of the advanced PCa was performed. Materials and Methods: Germline DDR gene mutations were assessed prospectively in DNA samples from 139 patients, using a five-gene panel (BRCA1, BRCA2, ATM, CHEK2, and NBN) targeted next-generation sequencing. Results: This study revealed an almost three-fold higher risk of localized PCa among mutation carriers as compared to non-carriers (OR 2.84 and 95% CI: 0.75-20.23, p = 0.16). The prevalence of germline DDR gene mutations in PCa cases was 16.8% (18/107) and they were detected only in cases with PI-RADS 4/5 lesions. BRCA1/BRCA2/ATM mutation carriers were 2.6 times more likely to have a higher (>1) cISUP grade group compared to those with a CHEK2 mutation (p = 0.27). However, the number of cISUP > 1-grade patients with a CHEK2 mutation was significantly higher in advanced PCa than in localized PCa: 66.67% vs. 23.08% (p = 0.047). Conclusions: The results of our study suggest the potential of genetic screening for selected DDR gene mutations for early identification of cases at risk of aggressive PCa.

Urinary microRNAs can predict response to abiraterone acetate in castration resistant prostate cancer: A pilot study.

BACKGROUND: Despite novel agents have been introduced to treat castration resistant prostate cancer (CRPC) during the last decade, up to one-third of CRPC patients face primary resistance to new generation compounds. Therefore, sensitive molecular tools are urgently needed for reliable treatment selection and response prediction. This study aimed to evaluate urinary miRNAs and blood circulating androgen receptor (AR) transcript level as a tool for noninvasive outcome prediction for CRPC patients undergoing abiraterone acetate (AA) therapy. METHODS: Prostate cancer-specific miR-148a, -365, -375, and -429 were analyzed in 129 urine samples collected from 100 CRPC patients before and during AA therapy via quantitative reverse transcription PCR. To test the prognostic value, urinary miRNA levels alone, as well as combined with AR level were associated with progression-free survival (PFS) and overall survival (OS). RESULTS: Level of urinary miR-375 was the highest in CRPC in comparison to noncancerous controls, as well as in combination with miR-429 was predictive for short PFS in AA-treated patients (HR = 2.2, 95% CI: 1.1-4.2, p = 0.023). Especially high prognostic power of all analyzed miRNAs was observed in CRPC cases with high blood AR levels. For PFS prediction a tandem of miR-429 and high AR reached HR of 5.0 (95% CI: 2.2-11.8, p < 0.001), while for prediction of OS the best combination was demonstrated by miR-148a and AR with HR of 3.1 (95% CI: 1.4-7.1, p = 0.006). CONCLUSIONS: Urinary miRNAs could be used as prognostic biomarkers for CRPC patients to predict response to AA therapy, especially for the cases with high blood AR levels.

The Emerging Role of Chromatin Remodeling Complexes in Ovarian Cancer.

Ovarian cancer (OC) is the fifth leading cause of women's death from cancers. The high mortality rate is attributed to the late presence of the disease and the lack of modern diagnostic tools, including molecular biomarkers. Moreover, OC is a highly heterogeneous disease, which contributes to early treatment failure. Thus, exploring OC molecular mechanisms could significantly enhance our understanding of the disease and provide new treatment options. Chromatin remodeling complexes (CRCs) are ATP-dependent molecular machines responsible for chromatin reorganization and involved in many DNA-related processes, including transcriptional regulation, replication, and reparation. Dysregulation of chromatin remodeling machinery may be related to cancer development and chemoresistance in OC. Some forms of OC and other gynecologic diseases have been associated with mutations in specific CRC genes. Most notably, ARID1A in endometriosis-related OC, SMARCA4, and SMARCB1 in hypercalcemic type small cell ovarian carcinoma (SCCOHT), ACTL6A, CHRAC1, RSF1 amplification in high-grade serous OC. Here we review the available literature on CRCs' involvement in OC to improve our understanding of its development and investigate CRCs as possible biomarkers and treatment targets for OC.

Urine Molecular Biomarkers for Detection and Follow-Up of Small Renal Masses.

Active surveillance (AS) is the best strategy for small renal masses (SRMs) management; however, reliable methods for early detection and disease aggressiveness prediction are urgently needed. The aim of the present study was to validate DNA methylation biomarkers for non-invasive SRM detection and prognosis. The levels of methylated genes TFAP2B, TAC1, PCDH8, ZNF677, FLRT2, and FBN2 were evaluated in 165 serial urine samples prospectively collected from 39 patients diagnosed with SRM, specifically renal cell carcinoma (RCC), before and during the AS via quantitative methylation-specific polymerase chain reaction. Voided urine samples from 92 asymptomatic volunteers were used as the control. Significantly higher methylated TFAP2B, TAC1, PCDH8, ZNF677, and FLRT2 levels and/or frequencies were detected in SRM patients' urine samples as compared to the control. The highest diagnostic power (AUC = 0.74) was observed for the four biomarkers panel with 92% sensitivity and 52% specificity. Methylated PCDH8 level positively correlated with SRM size at diagnosis, while TFAP2B had the opposite effect and was related to SRM progression. To sum up, SRMs contribute significantly to the amount of methylated DNA detectable in urine, which might be used for very early RCC detection. Moreover, PCDH8 and TFAP2B methylation have the potential to be prognostic biomarkers for SRMs.

Analysis of Intrinsic Breast Cancer Subtypes: The Clinical Utility of Epigenetic Biomarkers and TP53 Mutation Status in Triple-Negative Cases.

This study aimed at analyzing the DNA methylation pattern and TP53 mutation status of intrinsic breast cancer (BC) subtypes for improved characterization and survival prediction. DNA methylation of 17 genes was tested by methylation-specific PCR in 116 non-familial BRCA mutation-negative BC and 29 control noncancerous cases. At least one gene methylation was detected in all BC specimens and a 10-gene panel statistically significantly separated tumors from noncancerous breast tissues. Methylation of FILIP1L and MT1E was predominant in triple-negative (TN) BC, while other BC subtypes were characterized by RASSF1, PRKCB, MT1G, APC, and RUNX3 hypermethylation. TP53 mutation (TP53-mut) was found in 38% of sequenced samples and mainly affected TN BC cases (87%). Cox analysis revealed that TN status, age at diagnosis, and RUNX3 methylation are independent prognostic factors for overall survival (OS) in BC. The combinations of methylated biomarkers, RUNX3 with MT1E or FILIP1L, were also predictive for shorter OS, whereas methylated FILIP1L was predictive of a poor outcome in the TP53-mut subgroup. Therefore, DNA methylation patterns of specific genes significantly separate BC from noncancerous breast tissues and distinguishes TN cases from non-TN BC, whereas the combination of two-to-three epigenetic biomarkers can be an informative tool for BC outcome predictions.

A Systematic Review of Candidate Genes for Major Depression.

Background and Objectives: The aim of this systematic review was to analyse which candidate genes were examined in genetic association studies and their association with major depressive disorder (MDD). Materials and Methods: We searched PUBMED for relevant studies published between 1 July 2012 and 31 March 2019, using combinations of keywords: "major depressive disorder" OR "major depression" AND "gene candidate", "major depressive disorder" OR "major depression" AND "polymorphism". Synthesis focused on assessing the likelihood of bias and investigating factors that may explain differences between the results of studies. For selected gene list after literature overview, functional enrichment analysis and gene ontology term enrichment analysis were conducted. Results: 141 studies were included in the qualitative review of gene association studies focusing on MDD. 86 studies declared significant results (p < 0.05) for 172 SNPs in 85 genes. The 13 SNPs associations were confirmed by at least two studies. The 18 genetic polymorphism associations were confirmed in both the previous and this systematic analysis by at least one study. The majority of the studies (68.79 %) did not use or describe power analysis, which may have had an impact over the significance of their results. Almost a third of studies (N = 54) were conducted in Chinese Han population. Conclusion: Unfortunately, there is still insufficient data on the links between genes and depression. Despite the reported genetic associations, most studies were lacking in statistical power analysis, research samples were small, and most gene polymorphisms have been confirmed in only one study. Further genetic research with larger research samples is needed to discern whether the relationship is random or causal. Summations: This systematic review had summarized all reported genetic associations and has highlighted the genetic associations that have been replicated. Limitations: Unfortunately, most gene polymorphisms have been confirmed only once, so further studies are warranted for replicating these genetic associations. In addition, most studies included a small number of MDD cases that could be indicative for false positive. Considering that polymorphism loci and associations with MDD is also vastly dependent on interpersonal variation, extensive studies of gene interaction pathways could provide more answers to the complexity of MDD.

Distal Gastrectomy with Billroth II Reconstruction is Associated with Oralization of Gut Microbiome and Intestinal Inflammation: A Proof-of-Concept Study.

BACKGROUND: Subtotal gastrectomy with Billroth II reconstruction (SGB2) results in increased gastric pH and diminished gastric barrier. Increased gastric pH following PPI therapy has an impact on the gut microbiome, intestinal inflammation, and possibly patient health. If similar changes are present after SGB2, these can be relevant for patient health and long-term outcomes after surgery. The aim of the study is to investigate whether SGB2 is associated with specific changes in gut microbiome composition and intestinal inflammation. PATIENTS AND METHODS: This cross-sectional proof-of-concept study includes patients after SGB2 (n = 14) for early gastric cancer and their nongastrectomized in-house relatives as controls (n = 8). Fecal microbiome composition, intestinal inflammation (fecal calprotectin), gut permeability (DAO, LBP, sCD14), systemic inflammation (CRP) markers, and gastrointestinal symptoms are investigated. This study is registered at ClinicalTrials.gov (NCT03418428). RESULTS: Microbiome oralization following SGB2 was defined by an increase in Escherichia-Shigella, Enterococcus, Streptococcus, and other typical oral cavity bacteria (Veillonella, Oribacterium, and Mogibacterium) abundance. The fecal calprotectin was increased in the SGB2 group [100.9 (52.1; 292) vs. 25.8 (17; 66.5); p = 0.014], and calprotectin levels positively correlated with the abundance of Streptococcus (rs = 0.639; padj = 0.023). Gastrointestinal symptoms in SGB2 patients were associated with distinct taxonomic changes of the gut microbiome. CONCLUSIONS: SGB2 is associated with oralization of the gut microbiome; intestinal inflammation and microbiome changes were associated with gastrointestinal symptoms. These novel findings may open gut microbiome as a new target for therapy to improve quality of life and general patient health in long-term survivors after SGB2.

Epigenetic Biomarkers of Renal Cell Carcinoma for Liquid Biopsy Tests.

Renal cell carcinomas (RCC) account for 2-3% of the global cancer burden and are characterized by the highest mortality rate among all genitourinary cancers. However, excluding conventional imagining approaches, there are no reliable diagnostic and prognostic tools available for clinical use at present. Liquid biopsies, such as urine, serum, and plasma, contain a significant amount of tumor-derived nucleic acids, which may serve as non-invasive biomarkers that are particularly useful for early cancer detection, follow-up, and personalization of treatment. Changes in epigenetic phenomena, such as DNA methylation level, expression of microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), are observed early during cancer development and are easily detectable in biofluids when morphological changes are still undetermined by conventional diagnostic tools. Here, we reviewed recent advances made in the development of liquid biopsy-derived DNA methylation-, miRNAs- and lncRNAs-based biomarkers for RCC, with an emphasis on the performance characteristics. In the last two decades, a mass of circulating epigenetic biomarkers of RCC were suggested, however, most of the studies done thus far analyzed biomarkers selected from the literature, used relatively miniature, local, and heterogeneous cohorts, and suffered from a lack of sufficient validations. In summary, for improved translation into the clinical setting, there is considerable demand for the validation of the existing pool of RCC biomarkers and the discovery of novel ones with better performance and clinical utility.

Promoter Methylation of PRKCB, ADAMTS12, and NAALAD2 Is Specific to Prostate Cancer and Predicts Biochemical Disease Recurrence.

The molecular diversity of prostate cancer (PCa) has been demonstrated by recent genome-wide studies, proposing a significant number of different molecular markers. However, only a few of them have been transferred into clinical practice so far. The present study aimed to identify and validate novel DNA methylation biomarkers for PCa diagnosis and prognosis. Microarray-based methylome data of well-characterized cancerous and noncancerous prostate tissue (NPT) pairs was used for the initial screening. Ten protein-coding genes were selected for validation in a set of 151 PCa, 51 NPT, as well as 17 benign prostatic hyperplasia samples. The Prostate Cancer Dataset (PRAD) of The Cancer Genome Atlas (TCGA) was utilized for independent validation of our findings. Methylation frequencies of ADAMTS12, CCDC181, FILIP1L, NAALAD2, PRKCB, and ZMIZ1 were up to 91% in our study. PCa specific methylation of ADAMTS12, CCDC181, NAALAD2, and PRKCB was demonstrated by qualitative and quantitative means (all p < 0.05). In agreement with PRAD, promoter methylation of these four genes was associated with the transcript down-regulation in the Lithuanian cohort (all p < 0.05). Methylation of ADAMTS12, NAALAD2, and PRKCB was independently predictive for biochemical disease recurrence, while NAALAD2 and PRKCB increased the prognostic power of multivariate models (all p < 0.01). The present study identified methylation of ADAMTS12, NAALAD2, and PRKCB as novel diagnostic and prognostic PCa biomarkers that might guide treatment decisions in clinical practice.

Vitamin D and VDR Gene Polymorphisms' Association with Rheumatoid Arthritis in Lithuanian Population.

Background and Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune, multi-factorial disease, in which environmental and genetic factors play a major role. RA is possibly linked to vitamin D deficiency and vitamin D receptor (VDR) gene polymorphisms, and research demonstrates that FokI variant susceptibility is associated with increased disease risk among Caucasians. The aim of this study was to evaluate vitamin D deficiency prevalence and its correlation to RA clinical parameters, and to determine the possible association of VDR gene polymorphisms and RA susceptibility in the Lithuanian population. Materials and Methods: Overall, 206 RA patients and 180 age- and sex-matched healthy controls were enrolled at Vilnius University Hospital Santaros Klinikos after informed consent was obtained. The disease activity score 28 C-reactive protein (DAS28 CRP), rheumatoid arthritis impact of disease (RAID) score, and health assessment questionnaire (HAQ) were recorded in RA patients, and 25(OH)D serum levels were evaluated by chemiluminescent microparticle immunoassay for all subjects. Four VDR gene polymorphisms, BsmI, FokI, ApaI, and TaqI, were assessed using real-time PCR instruments and genotyping assays in both groups. Results: The study registered a high prevalence of 25(OH)D deficiency (<50 nmol/L) in RA patients (61.55% (n = 127)). The mean serum concentration in RA patients (44.96 ± 21.92 (nmol/L)) was significantly lower than in the healthy controls (54.90 ± 22.82 (nmol/L)), p < 0.0001. A significant inverse correlation between vitamin D level, DAS28 CRP, and HAQ scores was confirmed in RA patients, with p < 0.05. Still, there was no significant association between the overall risk of RA disease for any allele or genotype of the four VDR loci tested. Conclusions: The study confirmed that vitamin D deficiency is prevalent among RA patients and the 25(OH)D level is significantly lower compared with healthy controls. Lower vitamin D concentration was related with increased disease activity and disability scores. However, genetic analysis of four VDR polymorphisms did not confer the susceptibility to RA in Lithuanian population.

Salinomycin and dichloroacetate synergistically inhibit Lewis lung carcinoma cell proliferation, tumor growth and metastasis.

Drug combination is considered to be the cornerstone of cancer treatment. Simultaneous administration of two or more drugs but at lower doses not only increases cytotoxic effects on tumor cells, but also reduces side effects and possibly overcomes drug resistance. Salinomycin is a well-known cancer stem cell killer, and dichloroacetate is a pyruvate dehydrogenase kinase inhibitor that exclusively targets cells with altered mitochondrial activity, a characteristic being common to most of the cancer cells. In our recent study, we have demonstrated that salinomycin exerted a cytotoxic effect on colorectal carcinoma cells in the 2D and 3D cultures and provided evidence that the mechanism of their synergy was mediated by dichloroacetate-dependent inhibition of the activity of multidrug resistance proteins. In the current work, we confirmed the synergistic cytotoxic properties of salinomycin and dichloroacetate in the 2D and 3D cultures of Lewis lung carcinoma (LLC1) cells. To verify if a synergistic effect of these compounds persisted in vivo, we performed series of experiments using a syngeneic LLC1-C57BL/6 mouse model and demonstrated that combination therapy with salinomycin and DCA increased the survival rate of allografted mice, inhibited metastatic site formation and reduced the populations of cancer stem cells as well as cells that underwent the epithelial-to-mesenchymal transition. Our results demonstrate that a synergistic effect of salinomycin and dichloroacetate exists not only in vitro but also in vivo and suggest their benefits in the treatment of metastatic cancers.

Analysis of AR-FL and AR-V1 in Whole Blood of Patients with Castration Resistant Prostate Cancer as a Tool for Predicting Response to Abiraterone Acetate.

PURPOSE: Reliable molecular diagnostic tools are still unavailable for making informed treatment decisions and monitoring the response in patients with castration resistant prostate cancer. We evaluated the significance of whole blood circulating androgen receptor transcripts of full length (AR-FL) and splice variants (AR-V1, AR-V3 and AR-V7) as biomarkers of abiraterone acetate treatment resistance in patients with castration resistant prostate cancer. MATERIALS AND METHODS: After retrospective analysis in 112 prostate specimens AR-FL, AR-V1, AR-V3 and AR-V7 were evaluated in 185 serial blood samples, prospectively collected from 102 patients with castration resistant prostate cancer before and during abiraterone acetate therapy via reverse transcription quantitative polymerase chain reaction. RESULTS: AR-FL was present in all samples while AR-V1, AR-V3, AR-V7 and at least 1 of them was detected in 17%, 55%, 65% and 81% of castration resistant prostate cancer blood samples, respectively. The highest amount of AR-V1 was found in blood of patients whose response time was short and medium in comparison to extended. Patients with a higher level of AR-FL and/or AR-V1 had the shortest progression-free survival and overall survival (p <0.0001). CONCLUSIONS: Blood circulating AR-FL or AR-V1 can serve as blood based biomarkers for identification of the primary resistance to abiraterone acetate and the tool to monitor de novo resistance development during abiraterone acetate treatment.

Frequent DNA methylation changes in cancerous and noncancerous lung tissues from smokers with non-small cell lung cancer.

Cancer deaths account for nearly 10 million deaths worldwide each year, with lung cancer (LCa) as the leading cause of cancer-related death. Smoking is one of the major LCa risk factors, and tobacco-related carcinogens are potent mutagens and epi-mutagens. In the present study, we aimed to analyse smoking-related epigenetic changes in lung tissues from LCa cases. The study cohort consisted of paired LCa and noncancerous lung tissues (NLT) from 104 patients, 90 of whom were smokers or ex-smokers (i.e. ever smokers) at the time of diagnosis. DNA methylation status of tumour suppressor genes DAPK1, MGMT, p16, RASSF1 and RARB was screened by means of methylation-specific PCR (MSP) and further analysed quantitatively by pyrosequencing. Methylation of at least one gene was detected in 59% (61 of 104) of LCa samples and in 39% (41 of 104) of NLT. DAPK1 and RASSF1 were more frequently methylated in LCa than in NLT (P = 0.022 and P = 0.041, respectively). The levels of DNA methylation were higher in LCa than NLT at most of the analysed CpG positions. More frequent methylation of at least one gene was observed in LCa samples of ever smokers (63%, 57 of 90) as compared with never smokers (36%, 5 of 14; P = 0.019). In the ever smokers group, methylation of the genes also occurred in NLT, but was rare or absent in the samples of never smokers. Among the current smokers, RASSF1 methylation in LCa showed association with the number of cigarettes smoked per day (P = 0.017), whereas in NLT it was positively associated with the duration of smoking (P = 0.039). Similarly, p16 methylation in LCa of current smokers correlated with the larger number of cigarettes smoked per day (P = 0.047). Overall, DNA methylation changes were present in both cancerous and noncancerous tissues of LCa patients and showed associations with smoking-related parameters.

The Impact of Prostate Cancer Upgrading and Upstaging on Biochemical Recurrence and Cancer-Specific Survival.

Background and Objectives: Significant numbers of prostate cancer (PCa) patients experience tumour upgrading and upstaging between prostate biopsy and radical prostatectomy (RP) specimens. The aim of our study was to investigate the role of grade and stage increase on surgical and oncological outcomes. Materials and Methods: Upgrading and upstaging rates were analysed in 676 treatment-naïve PCa patients who underwent RP with subsequent follow-up. Positive surgical margin (PSM), biochemical recurrence (BCR), metastasis-free survival (MFS), overall (OS) and cancer specific survival (CSS) were analysed according to upgrading and upstaging. Results: Upgrading was observed in 29% and upstaging in 22% of PCa patients. Patients undergoing upgrading or upstaging were 1.5 times more likely to have a PSM on RP pathology. Both upgrading and upstaging were associated with increased risk for BCR: 1.8 and 2.1 times, respectively. Mean time to BCR after RP was 2.1 years in upgraded cases and 2.7 years in patients with no upgrading (p <0.001), while mean time to BCR was 1.9 years in upstaged and 2.8 years in non-upstaged cases (p <0.001). Grade and stage increase after RP were associated with inferior MFS rates and ten-year CSS: 89% vs. 98% for upgrading (p = 0.039) and 87% vs. 98% for upstaging (p = 0.008). Conclusions: Currently used risk stratification models are associated with substantial misdiagnosis. Pathological upgrading and upstaging have been associated with inferior surgical results, substantial higher risk of BCR and inferior rates of important oncological outcomes, which should be considered when counselling PCa patients at the time of diagnosis or after definitive therapy.