The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.

The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions.

Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identify HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyzes and single-cell RNA-sequencing demonstrate that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggest that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that is required for the gene expression program for pachytene transition during meiotic prophase in males.

The interplay between Wnt signaling pathways and microtubule dynamics.

Wnt signaling pathways represent an evolutionarily highly conserved, intricate network of molecular interactions that regulates various aspects of cellular behavior, including embryonic development and tissue homeostasis. Wnt signaling pathways share the ß-catenin-dependent (canonical) and the multiple ß-catenin-independent (non-canonical) pathways. These pathways collectively orchestrate a wide range of cellular processes through distinct mechanisms of action. Both the ß-catenin-dependent and ß-catenin-independent pathways are closely intertwined with microtubule dynamics, underscoring the complex crosstalk between Wnt signaling and the cellular cytoskeleton. This interplay involves several mechanisms, including how the components of Wnt signaling can influence the stability, organization, and distribution of microtubules. The modulation of microtubule dynamics by Wnt signaling plays a crucial role in coordinating cellular behaviors and responses to external signals. In this comprehensive review, we discussed the current understanding of how Wnt signaling and microtubule dynamics intersect in various aspects of cellular behavior. This study provides insights into our understanding of these crucial cellular processes.

Radiological complete response with regorafenib for multiple lung metastases of ascending colon cancer: a case report.

BACKGROUND: Regorafenib is an oral diphenylurea multikinase inhibitor and currently approved for use following third-line therapy for metastatic colorectal cancer (CRC) patients. Only one case has previously been reported of metastatic CRC showing a complete response (CR) to regorafenib. CASE PRESENTATION: A 68-year-old Japanese man underwent laparoscopy-assisted ileocecal resection and D3 lymphadenectomy due to his ascending colon cancer. Eighteen months after surgery, a laparoscopic hepatic left lateral segmentectomy was performed due to a liver tumor, and a pathological diagnosis of colorectal liver metastasis was made. Three months after the second surgery, contrast-enhanced computed tomography (CT) revealed multiple lung metastases. The patient had undergone 18 courses of the FOLFOX + bevacizumab chemotherapy regimen as their first-line therapy and 11 courses of the FOLFIRI + ramucirumab chemotherapy regimen as their second-line therapy. As their third-line therapy, the patient was administered the regorafenib chemotherapy regimen. We evaluated the chemotherapy treatment's effect on the lung tumors by CT after 3, 7, 11, and 17 courses of the regorafenib chemotherapy regimen, finding that the lung tumors had shrunk with time; thus, each tumor was considered a partial response (PR) based on the RECIST guidelines. After 21 courses of the regorafenib chemotherapy regimen, the chemotherapy was discontinued in response to the patient's wishes. Even at 1 and 3 months after the discontinuation of the chemotherapy, CT revealed that the lung tumors had shrunk, with each considered a PR. Furthermore, 9 months after the discontinuation of the chemotherapy, CT revealed scarring of the lung tumors. This was considered a CR, rather than a PR. The patient remains disease-free 18 months after the discontinuation of chemotherapy. CONCLUSIONS: In this paper, we present the second case of radiological CR with regorafenib for multiple lung metastases of ascending colon cancer. Currently, there is no consensus on a treatment strategy for patients with radiological CR.

Abscopal effect with fever of unknown cause during radiotherapy: Two case reports and review of the literature.

The abscopal effect is a rare phenomenon that is defined as regression of tumor lesions distant from irradiation targets. At our department, two cases with an abscopal effect with fever of unknown cause (FUC) and an inflammatory response during radiotherapy were encountered. Radiotherapy is a local treatment; therefore, it rarely causes systemic side effects during radiotherapy, and if a patient develops a fever during radiotherapy, it is frequently considered tumor fever. We experienced 2 cases of FUC during irradiation followed by abscopal effect. The obvious relationship between the abscopal effect and the fever remains to be clarified. However, FUC during radiotherapy may be a hint to the abscopal effect, considering that immune response and cytokines are closely related to the abscopal effect.

Reconsideration of Temperature Determined by the Excited-State Population Distribution of Hydrogen Atoms Based on Tsallis Entropy and Its Statistics in Hydrogen Plasma in Non-Equilibrium State.

In non-equilibrium plasmas, the temperature cannot be uniquely determined unless the energy-distribution function is approximated as a Maxwell-Boltzmann distribution. To overcome this problem, we applied Tsallis statistics to determine the temperature with respect to the excited-state populations in non-equilibrium state hydrogen plasma, which enables the description of its entropy that obeys q-exponential population distribution in the non-equilibrium state. However, it is quite difficult to apply the q-exponential distribution because it is a self-consistent function that cannot be solved analytically. In this study, a self-consistent iterative scheme was adopted to calculate q-exponential distribution using the similar algorithm of the Hartree-Fock method. Results show that the excited-state population distribution based on Tsallis statistics well captures the non-equilibrium characteristics in the high-energy region, which is far from the equilibrium-Boltzmann distribution. The temperature was calculated using the partial derivative of entropy with respect to the mean energy based on Tsallis statistics and using the coefficient of q-exponential distribution. An analytical expression was derived and compared with Boltzmann statistics, and the distribution was discussed from the viewpoint of statistical physics.

Risk-Adjusted Assessment of the Learning Curve for Pure Laparoscopic Donor Hepatectomy for Adult Recipients.

BACKGROUND: Studies on pure laparoscopic donor hepatectomy (PLDH) have been reported. However, only few studies have reported on the learning curve of PLDH. In this report, we aimed to determine the learning curve of PLDH in adult patients using cumulative sum (CUSUM) and risk-adjusted CUSUM (RA-CUSUM) analyses. METHODS: The data of donors who underwent PLDH at a single center between December 2012 and May 2022 were retrospectively reviewed. The learning curve was evaluated using the CUSUM and RA-CUSUM methods based on surgery duration. RESULTS: Forty-eight patients were finally included in the present study. The mean operation time was 393.6 ± 80.3 min. PLDH was converted to laparotomy in three cases (6.3%). According to the Clavien-Dindo classification, nine cases (18.8%) had higher-than-grade III postoperative complications and the most frequent complications were biliary complications. The CUSUM graph shows two peaks, at the 13th and 27th case. The multivariate analysis revealed that a body mass index ≥ 23 kg/m2 and intraoperative cholangiography were the only factors that were independently associated with longer operation time. Based on these results, an RA-CUSUM analysis was performed to assess the learning curve, which showed a decrease in the learning curve after 33 to 34 PLDH procedures. CONCLUSIONS: A learning curve effect was demonstrated in this study after 33 to 34 PLDH procedures. There are relatively many biliary complications, and it is necessary to further examine the method of bile duct transection.

A case of pathological complete response with liposomal irinotecan + 5-FU/LV for unresectable locally advanced pancreatic cancer.

BACKGROUND: Pancreatic cancer has one of the worst prognoses of any all cancers. 5-FU/leucovorin + irinotecan + oxaliplatin (FOLFIRINOX), gemcitabine (GEM) plus nab-paclitaxel regimens have been recognized as global-standard, first-line treatments for patients with advanced pancreatic cancer. The liposomal irinotecan (nal-IRI) + 5-FU/LV regimen is now included in treatment guidelines as a recommended and approved option for use in patients with metastatic pancreatic cancer that has progressed after GEM-based therapy and who have a suitable performance status and comorbidity profile. There is no report that nal-IRI + 5-FU/LV regimen was significantly effective, and we will report it because we experienced this time. CASE PRESENTATION: A 69-year-old man presented with epigastric pain, and a contrast computed tomography (CT) revealed an enhanced mass lesion measuring 33 × 27 mm on the pancreatic body with encasement of the common hepatic artery (CHA) and the splenic vein. An endoscopic ultrasound-guided fine needle aspiration was performed and demonstrated cytology consistent with adenocarcinoma. Therefore, we diagnosed the patient with unresectable locally advanced pancreatic cancer. The patient received the GEM and S-1 regimen; however, the adverse event was relatively severe. Then, 11 cycles of nal-IRI + 5-FU/LV regimen were administered. A CT scan revealed that the tumor had shrunk to 18 × 7 mm in diameter with encasement of the CHA. The encasement of the splenic vein had disappeared, without any distant metastases. From this post-chemotherapy evaluation and intraoperative frozen section of around the celiac artery, gastroduodenal artery and pancreas stump confirmed absence of tumor cells, we performed distal pancreatectomy with celiac axis resection. A histological examination of the surgical specimen revealed no evidence of residual adenocarcinoma, consistent with a pathological complete response to treatment. CONCLUSIONS: We present the first case of a pathological complete response with nal-IRI + 5-FU/LV for unresectable, locally advanced pancreatic cancer. In the future, nal-IRI may become a key drug for pancreatic cancer treatment.

Pure Laparoscopic Left Hepatectomy for Regrowth of Mucinous Cystic Neoplasm of the Liver after Laparoscopic Deroofing.

Background: Hepatic cystic lesions are common entities, most of which are simple hepatic cysts (SHCs). Mucinous cystic neoplasm of the liver (MCN-L) is a rare tumor characterized by ovarian-like stroma and accounts for <5% of all hepatic cysts. Distinguishing between SHCs and MCN-L is challenging because of the similarity in their imaging findings. Herein, we report a rare regrowth case of MCN-L after laparoscopic deroofing, treated with pure laparoscopic left hepatectomy. Case Presentation. A 63-year-old woman with a large hepatic cystic lesion and abdominal pain was referred to our hospital for surgical treatment. Computed tomography (CT) showed cystic lesions with septations arising from macrolobulations in the left medial segment. She underwent laparoscopic deroofing based on the diagnosis of SHCs; however, the final histopathological diagnosis was MCN-L. She chose observational follow-up, and MCN-L regrowth was detected on follow-up CT 6 months after the laparoscopic deroofing. We performed pure laparoscopic left hepatectomy for complete resection of the MCN-L. She had an uneventful postoperative course and no recurrence at the 5-year follow-up after the radical resection of the MCN-L. Conclusion: MCN-L is a rare entity, and accurate diagnosis with imaging modalities is greatly challenging. Laparoscopic hepatectomy for MCN-L should be considered as a strong alternative to secure safety and curability.

Mobilization of multilineage-differentiating stress-enduring cells into the peripheral blood in liver surgery.

PURPOSE: This study investigated whether liver damage severity relates to the mobilization of multilineage-differentiating stress-enduring (Muse) cells, which are endogenous reparative pluripotent stem cells, into the peripheral blood (PB) and whether the degree of mobilization relates to the recovery of liver volume following human liver surgery. METHODS: Forty-seven patients who underwent liver surgery were included in the present study. PB-Muse cells were counted before surgery, on postoperative days (PODs) 3 and on POD 7. Liver volume was measured using computed tomography before and after surgery. RESULTS: The PB-Muse cell count increased after surgery. The number of PB-Muse cells before surgery was higher, but without statistical significance in the group with neoplasms than in the healthy group that included liver donors (p = 0.065). Forty-seven patients who underwent liver surgery were divided into major hepatic resection (MHR; hepatectomy of three or more segments according to the Couinaud classification, n = 22) and minor hepatic resection (mhr; hepatectomy of two segments or less according to the Couinaud classification, n = 25) groups. PB-Muse cells increased at high rates among MHR patients (p = 0.033). Except for complication cases, PB-Muse cells increased at higher rates in the group with advanced liver volume recovery (p = 0.043). The predictive impact of the rate of increase in PB-Muse cells on the recovery of liver volume was demonstrated by multivariate analysis (OR 11.0, p = 0.014). CONCLUSIONS: PB-Muse cell mobilization correlated with the volume of liver resection, suggesting that the PB-Muse cell number reflects the degree of liver injury. Given that the degree of PB-Muse cell mobilization was related to liver volume recovery, PB-Muse cells were suggested to contribute to liver regeneration, although this mechanism remains unclear.

Single-Port Laparoscopic Duodenojejunostomy Employing Semi-Kocherization for a Young Female with Superior Mesenteric Artery Syndrome.

Single-port laparoscopic duodenojejunostomy employing semi-Kocherization performed for a patient with superior mesenteric artery (SMA) syndrome is presented in this report. A 24-year-old woman missed meals due to work pressure, and her body weight decreased from 42 kg to 27 kg within 6 months. After this severe weight loss, she suffered from postprandial abdominal pain. An enhanced computed tomography revealed that the aortomesenteric angle was 11° (narrow), and the distance was short at 4.5 mm. Duodenography also revealed dilatation of the proximal duodenum. These findings led to a diagnosis of SMA syndrome, and we performed single-port laparoscopic duodenojejunostomy. We first dissected the fusion between the duodenum and transverse mesocolon, such as Kocherization, enough to mobilize the duodenum; this procedure was termed semi-Kocherization. A gauze was placed in the dissected space for a landmark from the transverse mesocolon side. We confirmed the gauze at the duodenum's lateral side, then opened the transverse mesocolon, and pulled the duodenum out. We performed side-to-side duodenojejunostomy. The postoperative course was unremarkable, and she gained 4 kg within 2 months of discharge. Semi-Kocherization is shown to be an effective technique to increase duodenal mobility for performing anastomosis, and single-port laparoscopic surgery can reduce wounds and increase cosmesis.

Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells.

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

Standardized single-incision plus one-port laparoscopic left lateral sectionectomy: a safe alternative to the conventional procedure.

PURPOSE: Laparoscopic left lateral sectionectomy (LLLS) is a feasible and safe procedure with a relatively smooth learning curve. However, single-incision LLLS requires extensive surgical experience and advanced techniques. The aim of this study is to report the standardized single-incision plus one-port LLLS (reduced port LLLS, RPLLLS) technique and evaluate its safety, feasibility, and effectiveness for junior surgeons. METHODS: Between January 2008 and November 2020, the clinical records of 49 patients who underwent LLLS, divided into the conventional LLLS (n = 37) and the RPLLLS group (n = 12), were retrospectively reviewed. The patient characteristics, pathologic results, and operative outcomes were evaluated. RESULTS: A history of previous abdominal surgery in the RPLLLS group was significantly high (56.8% vs. 91.7%, p = 0.552). Notably, junior surgeons performed 62.2% of the conventional LLLSs and 58.4% of the standardized RPLLLSs. There were no significant differences between the two groups in terms of median operative time (121.0 vs. 113.5, p = 0.387), median blood loss (13.0 vs. 8.5, p = 0.518), median length of hospital stays (7.0 vs. 7.0, p = 0.408), and morbidity rate (2.7% vs. 0%, p = 0.565), respectively. CONCLUSION: This standardized RPLLLS is a feasible and safe alternative to conventional LLLS and may become the ideal training procedure for both junior surgeons and surgeons aiming to learn more complex procedures.

[Two Cases of Penoscrotal Strangulation].

Case 1 (42-year-old man) : The patient was examined for penoscrotal swelling that had continued for 1 month. An annular erosive skin ulcer was observed at the penoscrotal base, with distal swelling. Asking the patient about the history of his condition was difficult due to a history of mental illness. We suspected his symptoms were due to an embedded foreign object. As computed tomography indicated the presence of a subcutaneous foreign object, surgery was performed to remove it. A rubber band was found wrapped twice around the area. After releasing the strangulation, penoscrotal swelling improved. Case 2 (72-year-old man) : Penoscrotal swelling appeared after having an automobile tow hook attached to the penoscrotal base for 2 weeks. The patient was examined at the emergency room because he could not remove it on his own. A rescue squad was called, and they cut the strangulating object with an electric saw. After releasing the strangulation, penoscrotal swelling improved. Although we experienced 2 cases of penoscrotal strangulation involving strangulating objects with different characteristics, improvement was achieved in both by releasing the strangulation. The cases of penoscrotal strangulation reported in Japan with known strangulation type are reviewed.

Enterohaemorrhagic Escherichia coli O121:H19 acquired an extended-spectrum ß-lactamase gene during the development of an outbreak in two nurseries.

Enterohaemorrhagic Escherichia coli (EHEC) is an important human pathogen worldwide. Although serotype O157 is currently the most dominant and important EHEC strain, serotypes O26, O111, O91, O103 and O121 are also recognized as serious pathogens that affect public health. EHEC outbreaks often occur in nurseries and elderly care facilities. In 2012, a nursery outbreak of EHEC O121 occurred during which the bacterium acquired a plasmid-borne extended-spectrum ß-lactamase (ESBL) gene. ESBL-producing E. coli O86 was concurrently isolated from one of the EHEC patients. Therefore, we investigated the isolates by whole-genome sequence (WGS) analysis to elucidate the transmission dynamics of the EHEC strains and the ESBL plasmid. According to WGS-based phylogeny, all 17 EHEC O121 isolates were clonal, while E. coli O86 was genetically distant from the EHEC O121 isolates. The complete sequence of an ESBL plasmid encoding the CTX-M-55 ß-lactamase was determined using S1-PFGE bands, and subsequent mapping of the WGS reads confirmed that the plasmid sequences from EHEC O121 and E. coli O86 were identical. Furthermore, conjugation experiments showed that the plasmid was capable of conjugative transfer. These results support the hypothesis that EHEC O121 acquired an ESBL-producing plasmid from E. coli O86 during the outbreak. This report demonstrates the importance of implementing preventive measures during EHEC outbreaks to control both secondary infection and the spread of antimicrobial resistance factors.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the ß-catenin-dependent and ß-catenin-independent pathways. Wnt5a signaling through a ß-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

Vimentin-positive gastric adenocarcinoma arising in a hyperplastic polyp.

We report a case of vimentin-positive early gastric adenocarcinoma arising in a hyperplastic polyp (HP). A 72-year-old Japanese man was admitted for the detailed examination of a gastric polyp. He had a subtotal gastrectomy due to acute abdomen 12 years ago. Upper endoscopy revealed a pedunculated polyp measuring approximately 2 cm on the greater curvature of upper body of the remnant stomach. Magnifying endoscopy revealed that the microsurface pattern was irregular and partially absent accompanied with irregular microvessels at the upper end of the polyp. We speculated that the lesion was an adenocarcinoma arising in the HP. Endoscopic submucosal dissection (ESD) was performed. Histological examination of the ESD specimen revealed that the lesion consisted of well- to poorly differentiated adenocarcinoma at the protruding lesion and foveolar hyperplastic epithelia at the base of the polyp. Immunohistochemically, most of tumor cells that comprised poorly-differentiated adenocarcinoma were positive for both cytokeratin and vimentin. Although carcinomas have occasionally been found in HPs, the histological features of the present case are considered extremely unusual. To the best of our knowledge, this is the first case of vimentin-positive early gastric carcinoma arising in a HP.

Effects of various spacers between biotin and the phospholipid headgroup on immobilization and sedimentation of biotinylated phospholipid-containing liposomes facilitated by avidin-biotin interactions.

Immobilization and sedimentation of liposomes (lipid vesicles) are used in liposome-protein binding assays, facilitated by avidin/streptavidin/NeutrAvidin and biotinylated phospholipid-containing liposomes. Here, we examined the effects of three spacers [six-carbon (X), polyethylene glycol (PEG) 180 (molecular weight 180) and PEG2000 (molecular weight 2,000)] between biotin and the phospholipid headgroup on the immobilization and sedimentation of small unilamellar liposomes/vesicles (SUVs). PEG180 and PEG2000 showed more efficient immobilization of biotinylated SUVs on NeutrAvidin-coated plates than X, but X and PEG180 showed more efficient sedimentation of biotinylated SUVs upon NeutrAvidin addition than PEG2000. Thus, the most appropriate spacers differed between immobilization and sedimentation. A spacer for biotinylated SUVs must be selected according to the particular liposome-protein binding assays examined.

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

INTRODUCTION AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.