TP53 codon 72 polymorphism is associated with FGFR3 and RAS mutation in non-muscle-invasive bladder cancer

ObjectiveTP53, a well-known tumor-suppressor gene in bladder carcinogenesis, has a functional single-nucleotide polymorphism on codon 72. The aim of this study was to elucidate the association between TP53 codon 72 polymorphism and somatic mutations in bladder cancer.Material and methodsGermline TP53 codon 72 polymorphism and somatic mutations of 50 cancer-associated genes were analyzed in 103 bladder cancer patients (59 non-muscle-invasive and 44 muscle-invasive), using Taqman genotyping assay and target sequencing, respectively. The expression of FGF-FGFR signaling pathway genes was analyzed by RNA sequencing of frozen tissue.ResultsThe allele frequency of TP53 codon 72 in our cohort was 37, 42, and 21% for Arg/Arg, Arg/Pro, and Pro/Pro, respectively. Interestingly, the prevalence of FGFR3 mutation was higher in patients with the Arg allele, whereas that of the RAS mutation was higher in patients without the Arg allele. The same association was seen in non-muscle-invasive bladder cancer (NMIBC) patients and no differences were observed in muscle-invasive bladder cancer patients. In NMIBC, FGFR1 expression was higher in patients without the Arg allele and FGFR3 expression was higher in patients with the Arg allele.ConclusionThe germline TP53 codon 72 polymorphism was associated with mutations of FGFR3 or RAS and expression of FGFR1 and FGFR3 in NMIBC. These findings provide new insight into the molecular mechanisms underlying the influence of the genetic background on carcinogenesis in bladder cancer

An Environmental DNA Survey on Distribution of an Endangered Salmonid Species, Parahucho perryi, in Hokkaido, Japan

For protecting endangered species, precise understanding of their distribution is crucial. However, it is often very difficult to estimate at a large scale with conventional methods (e.g., casting nets or electrofishing for aquatic species) because of their low densities in the wild. Sakhalin taimen (Parahucho perryi) is one of the largest and most critically endangered freshwater salmonid fishes in the world. In this study, we applied an environmental DNA (eDNA) detection system for this species to 120 rivers in Hokkaido, the second largest main island of Japan. We successfully detected eDNA from Sakhalin taimen in seven rivers (5.8%). Although these rivers were widely distributed across the island, > 95% of the total amounts of eDNA were detected from region-A and -I, indicating that local populations in the other regions of Hokkaido are very small and on the brink of extinction. In addition, principal component analyses based on the eDNA-based estimation of Sakhalin taimen distribution and GIS revealed their distribution determinants including limited topographic relief of watershed as well as presence of wetlands and lagoons. Our results suggest that eDNA-based detection systems are an efficient means of monitoring the population status of endangered freshwater species at large scales

Projection range of eDNA analysis in marshes: a suggestion from the Siberian salamander (Salamandrella keyserlingii) inhabiting the Kushiro marsh, Japan

Background: Freshwater ecosystems are rapidly declining. The Siberian salamander (Salamandrella keyserlingii) which inhabits the Kushiro marsh in Hokkaido, Japan has lost some habitat due to human activity. There are many challenges associated with conventional monitoring methods, including cost, the need for specialist personnel, environmental impact, and ability to detect the presence of this species; thus, we investigated the feasibility of using environmental DNA (eDNA) analysis to detect its presence and identify its breeding grounds. Methods: We performed tank experiments to confirm eDNA emission from egg sacs, larvae, and adult Siberian salamanders in the water. We also performed water sampling and visual observation of egg sacs in the Kushiro marsh during the end of the breeding season and the larval season. Results: The tank experiments found eDNA emission from all growth stages. It also implied concentrated emissions just after spawning and after hatching, and limited emissions during the incubation phase in egg sacs. We also detected eDNA in the field, likely reflecting the distribution of egg sacs or larvae. Combining this data with visual observations, it was determined that the eDNA results from the field were best explained by the number of egg sacs within 7-10 m of the sampling point. Conclusions: The results of this investigation show that the breeding sites and habitats of marshland species can successfully be monitored using eDNA analysis. They also suggest that the eDNA results from the marshes may reflect the biomass that is in close range to the sampling point. These results support the increased use of eDNA analysis in marshes and provide knowledge that could improve the interpretation of future results

TP53 codon 72 polymorphism is associated with FGFR3 and RAS mutation in non-muscle-invasive bladder cancer.

ObjectiveTP53, a well-known tumor-suppressor gene in bladder carcinogenesis, has a functional single-nucleotide polymorphism on codon 72. The aim of this study was to elucidate the association between TP53 codon 72 polymorphism and somatic mutations in bladder cancer.Material and methodsGermline TP53 codon 72 polymorphism and somatic mutations of 50 cancer-associated genes were analyzed in 103 bladder cancer patients (59 non-muscle-invasive and 44 muscle-invasive), using Taqman genotyping assay and target sequencing, respectively. The expression of FGF-FGFR signaling pathway genes was analyzed by RNA sequencing of frozen tissue.ResultsThe allele frequency of TP53 codon 72 in our cohort was 37, 42, and 21% for Arg/Arg, Arg/Pro, and Pro/Pro, respectively. Interestingly, the prevalence of FGFR3 mutation was higher in patients with the Arg allele, whereas that of the RAS mutation was higher in patients without the Arg allele. The same association was seen in non-muscle-invasive bladder cancer (NMIBC) patients and no differences were observed in muscle-invasive bladder cancer patients. In NMIBC, FGFR1 expression was higher in patients without the Arg allele and FGFR3 expression was higher in patients with the Arg allele.ConclusionThe germline TP53 codon 72 polymorphism was associated with mutations of FGFR3 or RAS and expression of FGFR1 and FGFR3 in NMIBC. These findings provide new insight into the molecular mechanisms underlying the influence of the genetic background on carcinogenesis in bladder cancer

Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.

Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples.The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors

<i>FGFR3</i> mutation and <i>FGFR3-TACC3</i> fusion status.

<p>(A) The heatmap shows the distribution of <i>FGFR3</i> mutations and <i>FGFR3-TACC3</i> fusions with respect to T stage and pathological grade. (B) Subgroup analysis of NMIBC by T stage.</p

A schematic figure explaining how bDNA-FISH works.

<p>A schematic figure explaining how bDNA-FISH works.</p