Characterization of KIF20B as a novel prognostic biomarker and therapeutic target for breast cancer.

Despite advances in treatment and early detection, breast cancer remains one of the most common types of cancer and is the second leading cause of cancer death after lung cancer in women. Therefore, there is an urgent need to develop new biomarkers and therapeutic targets for the treatment of breast cancer. Based on gene expression profiles and subsequent screening performed in a preliminary study, kinesin family member 20B (KIF20B) was selected as a candidate target molecule, because it was highly and frequently expressed in all subtypes of breast cancer and barely detected in normal tissues. Reverse transcription­quantitative PCR and western blotting revealed that KIF20B mRNA and protein expression levels were upregulated in most breast cancer cell lines but were scarcely expressed in normal mammary epithelial cells. Immunohistochemical staining of a tissue microarray showed that KIF20B was detected in 145 out of 251 (57.8%) breast cancer tissues. Strong KIF20B expression was significantly related to advanced pathological N stage. Moreover, patients with breast cancer and strong KIF20B expression exhibited a significantly worse prognosis than those with weak or negative KIF20B expression (P<0.0001, log­rank test). In multivariate analysis, strong expression was an independent prognostic factor for patients with breast cancer. Furthermore, knockdown of KIF20B expression by small interfering RNA inhibited breast cancer cell proliferation and induced apoptosis. In addition, Matrigel cell invasion assays revealed that the invasiveness of breast cancer cells was significantly decreased by KIF20B silencing. Since KIF20B is an oncoprotein that is strongly expressed in highly malignant clinical breast cancer and serves a pivotal role in breast cancer cell proliferation, survival and invasion, KIF20B could be considered a candidate biomarker for prognostic prediction and a potential molecular target for developing new therapeutics, such as small molecule inhibitors, for a wide variety of breast cancers.

Glutamine deficiency drives transforming growth factor-ß signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-ß (TGF-ß)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-ß-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-ß-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-ß-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-ß-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-ß signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Increased RUNX3 expression mediates tumor-promoting ability of human breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major stromal component of human breast cancers and often promote tumor proliferation, progression and malignancy. We previously established an experimental CAF (exp-CAF) cell line equipped with a potent tumor-promoting ability. It was generated through prolonged incubation of immortalized human mammary fibroblasts with human breast cancer cells in a tumor xenograft mouse model. RESULTS: Herein, we found that the exp-CAFs highly express Runt-related transcription factor 3 (RUNX3), while counterpart fibroblasts do not. In breast cancer patients, the proportion of RUNX3-positive stromal fibroblast-like cells tends to be higher in cancerous regions than in non-cancerous regions. These findings suggest an association of RUNX3 with CAF characteristics in human breast cancers. To investigate the functional role of RUNX3 in CAFs, the exp-CAFs with or without shRNA-directed knockdown of RUNX3 were implanted with breast cancer cells subcutaneously in immunodeficient mice. Comparison of the resulting xenograft tumors revealed that tumor growth was significantly attenuated when RUNX3 expression was suppressed in the fibroblasts. Consistently, Ki-67 and CD31 immunohistochemical staining of the tumor sections indicated reduction of cancer cell proliferation and microvessel formation in the tumors formed with the RUNX3-suppressed exp-CAFs. CONCLUSION: These results suggest that increased RUNX3 expression could contribute to the tumor-promoting ability of CAFs through mediating cancer cell growth and neoangiogenesis in human breast tumors.

Oncogenic epidermal growth factor receptor signal-induced histone deacetylation suppresses chemokine gene expression in human lung adenocarcinoma.

Epidermal growth factor receptor (EGFR)-mutated (mt) lung adenocarcinoma (LA) is refractory to immune checkpoint inhibitors (ICIs). However, the mechanisms have not been fully elucidated. CD8+ T cell infiltration was significantly lower in EGFR-mt than in EGFR-wild-type LA, which was associated with suppression of chemokine expression. Since this T cell-deserted tumor microenvironment may lead to the refractoriness of ICIs against EGFR-mt LA, we investigated the mechanism by focusing on the regulation of chemokine expression. The expression of C-X-C motif ligand (CXCL) 9, 10 and 11, which constitute a gene cluster on chromosome 4, was suppressed under EGFR signaling. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) revealed open chromatin peaks near this gene cluster following EGFR-tyrosine kinase inhibitor (TKI) treatment. The histone deacetylase (HDAC) inhibitor recovered the expression of CXCL9, 10 and 11 in EGFR-mt LA. Nuclear HDAC activity, as well as histone H3 deacetylation, were dependent on oncogenic EGFR signaling. Furthermore, the Cleavage Under Targets and Tagmentation (CUT & Tag) assay revealed a histone H3K27 acetylation peak at 15 kb upstream of CXCL11 after treatment with EGFR-TKI, which corresponded to one of the open chromatin peaks detected by ATAC-seq. The data suggest that EGFR-HDAC axis mediates silencing of the chemokine gene cluster through chromatin conformational change, which might be relevant to the ICI resistance by creating T cell-deserted tumor microenvironment. Targeting this axis may develop a new therapeutic strategy to overcome the ICI resistance of EGFR-mt LA.

Effects of a Nanophase-Separated Structure on Mechanical Properties and Proton Conductivity of Acid-Infiltrated Block Polymer Electrolyte Membranes under Non-Humidification.

Acid-infiltrated block polymer electrolyte membranes adopting a spherical or lamellar nanophase-separated structure were prepared by infiltrating sulfuric acid (H2SO4) into polystyrene-b-poly(4-vinylpyridine)-b-polystyrene (S-P-S) triblock copolymers to investigate the effects of its nanophase-separated structure on mechanical properties and proton conductivities under non-humidification. Lamellae-forming S-P-S/H2SO4 membranes with a continuous hard phase generally exhibited higher tensile strength than sphere-forming S-P-S/H2SO4 membranes with a discontinuous hard phase even if the same amount of Sa was infiltrated into each neat S-P-S film. Meanwhile, the conductivities of lamellae-forming S-P-S/H2SO4 membranes under non-humidification were comparable or superior to those of sphere-forming S-P-S/H2SO4 membranes, even though they were infiltrated by the same weight fraction of H2SO4. This result is attributed to the conductivities of S-P-S/H2SO4 membranes being greatly influenced by the acid/base stoichiometry associated with acid-base complex formation rather than the nanophase-separated structure adopted in the membranes. Namely, there are more free H2SO4 moieties that can release free protons contributing to the conductivity in lamellae-forming S-P-S/H2SO4 membranes than sphere-forming S-P-S/H2SO4, even when the same amount of H2SO4 was infiltrated into the S-P-S.

Genomic profiling amplifies the utility of endoscopic ultrasound-guided fine needle biopsy by identifying clinically applicable druggable mutations in pancreatic cancer.

BACKGROUND: Genomic profiling of tumors is available, but whether the small fragment obtained via endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) is sufficient for these examinations is unknown. Here we investigated whether EUS-FNB specimens are suitable for genomic profiling to identify oncogenic and drug-matched mutations. METHODS: We constructed a pancreatobiliary cancer panel for targeted panel sequencing that covered 60 significantly mutated genes and compared the results with those of whole-exome sequencing (WES). In total, 20 and 53 formalin-fixed paraffin-embedded tissues obtained via surgery and EUS-FNB were analyzed, respectively. First, we examined the DNA quality and genomic profiles of 20 paired samples from 20 malignant lesions obtained via surgery and EUS-FNB. We then tested 33 samples obtained via EUS-FNB from 24 malignant and 9 benign lesions for the discrimination of malignancy. Finally, we explored drug-matched mutations from EUS-FNB specimens. RESULTS: Although the DNA quantity obtained via surgery was higher than that obtained via EUS-FNB (P = 0.017), the DNA quality and mean depth were equivalent (P = 0.441 and P = 0.251). Panel sequencing of EUS-FNB specimens identified more oncogenic mutations than WES (90 % vs. 50 %). Furthermore, the number of oncogenic mutations did not differ between EUS-FNB and surgically resected specimens. Genomic profiling of EUS-FNB specimens enabled the discrimination of malignancy with 98 % accuracy. Of 44 malignant lesions, drug-matched alterations were identified in 14 % (6/44) of malignant lesions. CONCLUSION: EUS-FNB specimens can be widely utilized for diagnostic purposes, discrimination of malignancy, and detection of drug-matched mutations for the treatment of pancreatic cancer.

The Largest Quasicrystalline Tiling with Dodecagonal Symmetry from a Single Pentablock Quarterpolymer of the AB1CB2D Type.

A quasicrystalline tiling pattern with tile size of ca. 60 nm has been discovered in the bulk state of a four-component pentablock polymer molecule of the AS1IS2P type, where A, S, I, and P denote poly(4-vinylbenzyldimethylamine), polystyrene, polyisoprene, and poly(2-vinylpyridine), respectively. The polymer samples used were prepared by anionic polymerizations and have narrow molecular weight distribution. The sample films were obtained by an extremely slow solvent-cast process from dilute solutions of tetrahydrofuran for 14 days. It has been found by TEM observation that the quarterpolymer, AS1IS2P-4 (Mn = 149 kg/mol, ϕA/ϕS1/ϕI/ϕS2/ϕP = 0.12/0.27/0.20/0.29/0.13), reveals two final stable structures, i.e., a 3.3.4.3.4 periodic tiling pattern as a minor component and a quasicrystalline (QC) tiling with dodecagonal symmetry as a major component, where the former includes a triangle/square number ratio of 2 and the latter has one of approximately 2.28, which is close enough to the ideal ratio, 4/ 3 ≑ 2.31, for the triangle/square random tiling of the dodecagonal QC tiling (DDQC). Two structures were also clearly proved by SAXS diffraction patterns. Here, it should be noted this QC structure having a tile side length of ca. 60 nm was created with a single block polymer molecule.

Holliday junction recognition protein as a prognostic biomarker and therapeutic target for oral cancer.

Since oral cancer (OC) is highly malignant and the efficacy of standard treatments is limited, the development of new therapeutics is urgently awaited. To identify potential molecular targets for new OC diagnosis and therapies, we screened oncoantigens by gene expression profile and focused on Holliday junction recognition protein (HJURP), a mammalian centromere­specific chaperone. HJURP was found to be highly expressed in the majority of OC cell lines and tissues as compared to normal oral epithelial cells. Tissue microarray analysis confirmed that HJURP was expressed in 103 (67.8%) of 152 OC tissue specimens, but expression in normal oral tissues was limited. Positive HJURP expression was significantly correlated with shorter overall survival (P=0.003). Depletion of HJURP by small­interfering RNAs dramatically inhibited the growth of OC cells by inhibition of cell cycle progression and induced senescence of OC cells. In addition, inhibition of the interaction between HJURP and CENP­A significantly suppressed the growth of OC cells. These results indicate that HJURP is a potential prognostic biomarker, and targeting HJURP and its molecular pathway presents a new strategy for the development of treatments against OC.

Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer.

Oral cancer is a leading cause of cancer­related death worldwide. Current treatment for oral cancer includes surgery, radiotherapy, and chemotherapy; however, their effectiveness is still limited. To identify a new prognostic biomarker and therapeutic target for oral cancer, the Opa interacting protein 5 (OIP5), which plays an essential role in the proper segregation of chromosomes, was examined. Immunohistochemical staining using tissue microarrays indicated that OIP5 was expressed in 120 of 164 (73.2%) oral cancers but was minimally expressed in normal oral tissues. OIP5 expression was significantly associated with poor prognosis in patients with oral cancer. Overexpression of OIP5 enhanced the growth of oral cancer cells, whereas OIP5 knockdown using small interfering RNAs (siRNAs) significantly inhibited cell growth through cell cycle arrest at the G2/M phase. Suppression of OIP5 expression also induced senescence of oral cancer cells. Overall, the findings of the present study suggest that OIP5 may be a candidate prognostic biomarker and therapeutic target in oral cancer.

3D Culture Platform for Enabling Large-Scale Imaging and Control of Cell Distribution into Complex Shapes by Combining 3D Printing with a Cube Device.

While organoid differentiation protocols have been widely developed, local control of initial cell seeding position and imaging of large-scale organoid samples with high resolution remain challenging. 3D bioprinting is an effective method to achieve control of cell positioning, but existing methods mainly rely on the use of synthetic hydrogels that could compromise the native morphogenesis of organoids. To address this problem, we developed a 3D culture platform that combines 3D printing with a cube device to enable an unrestricted range of designs to be formed in biological hydrogels. We demonstrated the formation of channels in collagen hydrogel in the cube device via a molding process using a 3D-printed water-soluble mold. The mold is first placed in uncured hydrogel solution, then easily removed by immersion in water after the gel around it has cured, thus creating a mold-shaped gap in the hydrogel. At the same time, the difficulty in obtaining high-resolution imaging on a large scale can also be solved as the cube device allows us to scan the tissue sample from multiple directions, so that the imaging quality can be enhanced without having to rely on higher-end microscopes. Using this developed technology, we demonstrated (1) mimicking vascular structure by seeding HUVEC on the inner walls of helix-shaped channels in collagen gels, and (2) multi-directional imaging of the vascular structure in the cube device. Thus, this paper describes a concerted method that simultaneously allows for the precise control of cell positioning in hydrogels for organoid morphogenesis, and the imaging of large-sized organoid samples. It is expected that the platform developed here can lead to advancements in organoid technology to generate organoids with more sophisticated structures.

An in vitro system for experimentally induced cryptorchidism.

Cryptorchidism is one of the most common abnormalities of male sexual development, and is characterized by the failure of the testis to descend into the scrotum. Despite extensive studies of cryptorchidism over the past century, the mechanisms for temperature-induced germ-cell loss are not well understood. All of the main cell types in the testis are believed to be affected by the elevated testis temperature induced by cryptorchidism. The cooler temperature in the special environment of the scrotum is required for maintaining optional conditions for normal spermatogenesis. Many studies reported that experimentally induced cryptorchidism caused germ cell apoptosis and suppressed spermatogenesis. However, other factors including hormones must also be examined for cryptorchidism. To explore the mechanism for cryptorchidism, in vitro cultures of testes have been used, but complete spermatogenesis using in vitro methods was not accomplished until 2011. In 2011, Sato et al. (Nature, 471, 504-507) reported the in vitro production of functional sperm in cultured neonatal mouse testes. Using this in vitro system, for the first time, we report that spermatogenesis was abrogated at 37 °C, in accordance with in vivo surgery-mediated cryptorchidism, while spermatogenesis proceeded at 34 °C in cultured testes. This result clearly showed that temperature is the sole determinant of cryptorchidism. Moreover, we found that spermatogenesis was arrested before early spermatocytes at 37 °C. In conclusion, using our in vitro system, we have demonstrated that (1) temperature is the determining factor for cryptorchidism, and (2) higher temperature (37 °C) suppresses DNA synthesis in spermatogenesis.

Multiseptate gallbladder coexisting with pancreaticobiliary maljunction treated by laparoscopic cholecystectomy: report of a pediatric case.

BACKGROUND: A multiseptate gallbladder is a very rare congenital malformation in which the lumen is divided into variously sized multiseptal compartments. The pathogenesis and natural history of this disease remain uncertain. We herein describe a pediatric case of a multiseptate gallbladder with pancreaticobiliary maljunction (PBM), which was treated by laparoscopic cholecystectomy. CASE PRESENTATION: A 5-year-old girl was referred to our hospital, because a multiseptate gallbladder had been incidentally detected on abdominal ultrasonography when she presented for transient abdominal pain. Ultrasonography showed hyperechoic septa throughout the lumen of the gallbladder, giving it a honeycomb appearance. The atrophied gallbladder had weak or no contractility. Magnetic resonance cholangiopancreatography performed to detect other coexisting biliary disorders revealed PBM without dilatation of the common bile duct. Although physical examination and laboratory tests revealed no abnormalities, we performed laparoscopic cholecystectomy to prevent cholecystitis and reduce the risk of cancer secondary to the PBM. CONCLUSIONS: In recent pediatric case reports, the indication and timing of cholecystectomy has tended to be determined by the patient's symptoms and the presence of biliary complications. In the present case, however, the combination of a multiseptate gallbladder and PBM may become problematic in the future. Surgical treatment without delay was appropriate even in this pediatric patient.

Deep convolutional neural network-based algorithm for muscle biopsy diagnosis.

Histopathologic evaluation of muscle biopsy samples is essential for classifying and diagnosing muscle diseases. However, the numbers of experienced specialists and pathologists are limited. Although new technologies such as artificial intelligence are expected to improve medical reach, their use with rare diseases, such as muscle diseases, is challenging because of the limited availability of training datasets. To address this gap, we developed an algorithm based on deep convolutional neural networks (CNNs) and collected 4041 microscopic images of 1400 hematoxylin-and-eosin-stained pathology slides stored in the National Center of Neurology and Psychiatry for training CNNs. Our trained algorithm differentiated idiopathic inflammatory myopathies (mostly treatable) from hereditary muscle diseases (mostly non-treatable) with an area under the curve (AUC) of 0.996 and achieved better sensitivity and specificity than the diagnoses done by nine physicians under limited diseases and conditions. Furthermore, it successfully and accurately classified four subtypes of the idiopathic inflammatory myopathies with an average AUC of 0.958 and classified seven subtypes of hereditary muscle disease with an average AUC of 0.936. We also established a method to validate the similarity between the predictions made by the algorithm and the seven physicians using visualization technology and clarified the validity of the predictions. These results support the reliability of the algorithm and suggest that our algorithm has the potential to be used straightforwardly in a clinical setting.

Hexagonally Packed Cylindrical Structures with Multiple Satellites from Pentablock Quarterpolymers of the AB1CB2D Type and Their Blends with Homopolymers.

Three kinds of hexagonally packed cylindrical (HPC) structures with 10/8 satellites have been found from a neat pentablock quarterpolymer of the AS1IS2P type, composed of poly(4-vinylbenzyldimethylamine) (A), polystyrene (S), polyisoprene (I), and poly(2-vinylpyridine) (P), and block polymer/homopolymer blends. AS1IS2P-2 (M = 276k, ϕA/ϕS1/ϕI/ϕS2/ϕP = 0.08/0.17/0.12/0.33/0.30) shows HPC of P with 10 single-arrayed I satellites and 6 A subsatellites in the S matrix, where the I/P ratio number is 5, while AS1IS2P-2 (M = 325k, ϕA/ϕS1/ϕI/ϕS2/ϕP = 0.07/0.31/0.14/0.18/0.30)/Sh(M;11k) = 90/10 blend reveals another HPC of P with 10 I satellites, giving an I/P ratio of 6, whose array of I is a single/double mixture. Moreover, AS1IS2P-1/Sh/Ah(M;9k) = 90/5/5 blend represents two enantiomeric HPC packings with 8 double-arrayed I satellites having an I/P ratio of 7, which can be considered to be approximants of two enantiomeric 3.3.3.3.6 Archimedean tilings. These structures were conceived to be formed by the enhanced miscibility of tail A-chains with added homopolymers, resulting in an increase of the (A + S)/I interaction leading to the split of I domains.

Triply Helical Giant Domain with Homochirality in a Terpolymer Blend System.

Hexagonally packed coaxial triply helical domains with a mesoscopic length scale in matrices were created from an S1IS2P tetrablock terpolymer/Sh homopolymer blend system, wherein S1, S2, and Sh denote polystyrene, I is polyisoprene, and P represents poly(2-vinylpyridine). Two terpolymers, i.e., S1IS2P-3 (S1/I/S2/P = 0.50/0.17/0.19/0.14, M = 134k) and S1IS2P-4 (S1/I/S2/P = 0.58/0.16/0.10/0.16, M = 173k), were blended with Sh (M = 3k) at various concentrations. In the S1IS2P-3/Sh = 80/20 blend, the helical domain of P (o.d.= 19 nm; h.p. = 34 nm) was displayed by TEM, and the helical I phase (o.d. = 55 nm; i.d. = 29 nm; h.p. = 34 nm) was clearly demonstrated by 3D-TEM tomography. Essentially the same structure was confirmed to be created from the S1IS2P-4/Sh blend. These findings point out that S2 chains fill the gap between the I and P helices, and hence the intermediate S phase also has a helical nature. Moreover, it is worth noting that grains composed of hexagonally packed helices reveal homochirality.

Melt rheology of tadpole-shaped polystyrenes with different ring sizes.

In this study, linear melt rheology of a single-tail tadpole-shaped polystyrene, ST-30/80, having ring and linear sizes of MR ∼ 30 kg mol-1 and ML ∼ 80 kg mol-1, respectively, was examined, and the effect of the ring size on rheological properties of tadpole polymers was discussed by comparing with the data of the previously reported tadpole samples having MR ∼ 60 kg mol-1. ST-30/80 exhibits an entanglement plateau and shows a clearly slower terminal relaxation than that of its component ring and linear polymers. When the zero-shear viscosity η0 for ST-30/80 is plotted against the molecular weight of a linear tail chain, the data point lies on the single curve of η0 for 4- and 6-arm star polymers and the single-tail tadpoles with MR ∼ 60 kg mol-1. These results suggest that the tadpole molecule in this study spontaneously forms a characteristic entanglement network, i.e., the intermolecular ring-linear threading, in the same manner as the previous tadpole samples, even though the size of the ring part is just slightly larger than the entanglement molecular weight (i.e., MR ∼ 1.8Me).

Integrated On-Chip 3D Vascular Network Culture under Hypoxia.

We developed a portable device made of poly(dimethylsiloxane) (PDMS)/polymethylmethacrylate (PMMA) for long-term 3D cell culture of vascular endothelial cells for the development of a vascular network and evaluated the device under different transitions between normoxia and hypoxia with good optical accessibility. The combination of a nested reservoir device and a bicarbonate/ascorbate buffer system accomplished on-chip incubation with 4.91 ± 0.86% pO2 and 5.19 ± 1.70% pCO2 for up to 10 days. Seventy-two hours of normoxic incubation preceding hypoxic culture increased the cell viability, network formation, and size and stability of the resulting lumens compared with those completely maintained in normoxia for the same total duration. We employed different parameters of the network (e.g., total mesh area, total length, number of branches, among others) for the comparison of different oxygen treatments in the device. The differential effect of hypoxic conditions based on the maturity of the vessels may be used as an external factor to improve vascular development in vitro.

Characterization of KIF20A as a prognostic biomarker and therapeutic target for different subtypes of breast cancer.

The aim of the present study was to identify novel prognostic biomarkers and therapeutic targets for breast cancer; thus, genes that are frequently overexpressed in several types of breast cancer were screened. Kinesin family member 20A (KIF20A) was identified as a candidate molecule during this process. Immunohistochemical staining performed using tissue microarrays from 257 samples of different breast cancer subtypes revealed that KIF20A was expressed in 195 (75.9%) of these samples, whereas it was seldom expressed in normal breast tissue. KIF20A protein was expressed in all types of breast cancer observed. However, it was more frequently expressed in human epidermal growth factor receptor 2 (HER2)­positive and triple­negative breast cancer than in the luminal type. Moreover, KIF20A expression was significantly associated with the poor prognosis of patients with breast cancer. A multivariate analysis indicated that KIF20A expression was an independent prognostic factor for patients with breast cancer. The suppression of endogenous KIF20A expression using small interfering ribonucleic acids or via treatment with paprotrain, a selective inhibitor of KIF20A, significantly inhibited breast cancer cell growth through cell cycle arrest at the G2/M phase and subsequent mitotic cell death. These results suggest that KIF20A is a candidate prognostic biomarker and therapeutic target for different types of breast cancer.

Detection of neoantigen-reactive T cell clones based on the clonal expansion using next-generation sequencing of T cell receptor ß complementarity-determining region 3.

Development of mechanism-driven biomarkers for immune checkpoint inhibitors (ICIs) in cancer immunotherapy requires sensitive and efficacious assays to identify tumor antigen (Ag)-specific T cells. We demonstrated the concept for a sensitive method to determine Ag-reactive T cell clones based on clonal expansion using model neoantigens (NeoAgs) rather than cytokine production. Sequential increase in T cell clonal frequencies following Ag stimulation was detected by next generation sequencing (NGS) of T cell receptor ß (TCR ß) complementarity-determining region 3 (CDR3), with a higher sensitivity than that of enzyme-linked immunospot (ELISPOT) by 100-fold. The TCRß CDR3 sequences could represent useful markers to track dynamic changes during immunotherapy. The TCRß NGS-based method could represent a novel platform both for the development of new biomarkers as well as several therapeutic options.