Involvement of the TNF-α Pathway in TKI Resistance and Suggestion of TNFR1 as a Predictive Biomarker for TKI Responsiveness in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Mechanism and predictive biomarkers for tyrosine kinase inhibitor (TKI) resistance of advanced clear cell renal cell carcinoma (ccRCC) have not been fully evaluated. METHODS: We performed gene expression profiling on samples from an acquired TKI resistance cohort that consisted of 10 cases of TKI-treated ccRCC patients with matched tumor tissues harvested at pre-treatment and TKI-resistant post-treatment periods. In addition, a public microarray dataset from patient-derived xenograft model for TKI-treated ccRCC (GSE76068) was retrieved. Commonly altered pathways between the datasets were investigated by Ingenuity Pathway Analysis using commonly regulated differently expressed genes (DEGs). The significance of candidate DEG on intrinsic TKI resistance was assessed through immunohistochemistry in a separate cohort of 101 TKI-treated ccRCC cases. RESULTS: TNFRSF1A gene expression and tumor necrosis factor (TNF)-α pathway were upregulated in ccRCCs with acquired TKI resistance in both microarray datasets. Also, high expression (> 10% of labeled tumor cells) of TNF receptor 1 (TNFR1), the protein product of TNFRSF1A gene, was correlated with sarcomatoid dedifferentiation and was an independent predictive factor of clinically unfavorable response and shorter survivals in separated TKI-treated ccRCC cohort. CONCLUSION: TNF-α signaling may play a role in TKI resistance, and TNFR1 expression may serve as a predictive biomarker for clinically unfavorable TKI responses in ccRCC.

Epithelial-mesenchymal transition as a mechanism of resistance to tyrosine kinase inhibitors in clear cell renal cell carcinoma.

Tyrosine kinase inhibitors (TKIs) are widely accepted as treatment for metastatic clear cell renal cell carcinoma (ccRCC). However, most patients eventually experience disease progression despite TKI treatment, even if the initial response is favorable. To define the underlying mechanism of TKI resistance, 10 TKI-treated metastatic ccRCC cases in which tumor samples were harvested before treatment and immediately after disease progression were examined. Gene expression profiles and copy number variations of matched pre- and post-treatment tumor samples were investigated. Altered biologic characteristics were confirmed in sunitinib-resistant ccRCC cell lines, which were generated by long-term treatment with sunitinib-containing media. Gene transcript levels related to the cell cycle and epithelial-mesenchymal transition (EMT) were significantly upregulated in the treated tumor samples compared with the pre-treatment samples. The mitotic count and sarcomatoid component were significantly increased in treated tumor samples. Alteration of EMT-related genes was also demonstrated in a sunitinib-resistant ccRCC cell line that showed enhanced migration and invasion compared to the parent cell line. siRNA-induced inhibition of EMT-related gene expression significantly suppressed the migration and invasion capacity of TKI-resistant cell lines. The present study shows that both ccRCC cases that progressed after TKI treatment and sunitinib-resistant ccRCC cell lines demonstrated alteration of EMT-related gene expression and enhancement of EMT-related behavior. These results suggest that EMT may explain the aggressive behavior of TKI-resistant ccRCC.

Prognostic Significance of Macroscopic Appearance in Clear Cell Renal Cell Carcinoma and Its Metastasis-Predicting Model.

Prognostic significance of macroscopic appearance of clear cell renal cell carcinoma (ccRCC) has not yet been studied. This study aimed to define the prognostic significance of macroscopic appearance and to propose a prognostic model for post-operative metastasis in ccRCC. A total of 1,025 patients with ccRCC were analyzed for the development dataset. A separate cohort of 399 such patients was used as an external validation dataset. Macroscopic appearances were initially divided into four groups, but were later divided into two groups: a simple nodular group (700 cases, 68.3%) and an irregular outline group (325 cases, 32.7%). During the 66.1-month mean follow-up period, 98 patients (9.6%) developed metastasis. Univariate analysis revealed that metastasis was associated with older age, radical nephrectomy, larger tumor size, higher tumor grade and stage, and the irregular outline group. On multivariate analysis, age, tumor size, and macroscopic appearance remained as independent prognostic factors. These factors were used to build a prognostic model, which divided into three risk groups. The probabilities of 5-year metastasis-free survival in the low-, intermediate-, and high-risk groups were 98%, 83%, and 53%, respectively. The results showed prognostic significance of macroscopic appearance in ccRCC and propose a prognostic model to guide post-operative management of patients with ccRCC.

Sphingorhabdus arenilitoris sp. nov., isolated from a coastal sand, and reclassification of Sphingopyxis rigui as Sphingorhabdus rigui comb. nov. and Sphingopyxis wooponensis as Sphingorhabdus wooponensis comb. nov.

A Gram-stain-negative, strictly aerobic, non-flagellated and rod-shaped bacterial strain, designated GJR-7(T), was isolated from coastal sand of the South Sea of South Korea. Strain GJR-7(T) grew optimally at 30 °C, at pH 7.0-7.5 and without NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GJR-7(T) clustered with the type strains of Sphingopyxis wooponensis, Sphingopyxis rigui and Sphingorhabdus planktonica, with which it exhibited 16S rRNA gene sequence similarity values of 96.0-96.3%. Sequence similarities to the type strains of other recognized species were less than 95.5%. Strain GJR-7(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c, 11-methyl C(18 : 1)ω7c and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c) or C(14 : 0) 2-OH. The major polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and one unidentified glycolipid. The DNA G+C content of strain GJR-7(T) was 55.5 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, revealed that strain GJR-7(T) is separated from the type strains of Sphingopyxis wooponensis, Sphingopyxis rigui and Sphingorhabdus planktonica. On the basis of the data presented, strain GJR-7(T) is considered to represent a novel species of the genus Sphingorhabdus, for which the name Sphingorhabdus arenilitoris sp. nov. is proposed. The type strain is GJR-7(T) ( = KCTC 42051(T) = CECT 8531(T)). It is also proposed that Sphingopyxis wooponensis and Sphingopyxis rigui should be reclassified as members of the genus Sphingorhabdus.

Loktanella variabilis sp. nov., isolated from a tidal flat.

Two Gram-negative, aerobic and rod-shaped or ovoid bacterial strains with different colony colours (greyish yellow and moderate orange), designated J-MR2-Y(T) and J-MR2-O, were isolated from a tidal flat in the South Sea of South Korea. The two novel strains grew optimally at 35-37 °C. Strains J-MR2-Y(T) and J-MR2-O showed no difference in their 16S rRNA gene sequences, and the mean DNA-DNA relatedness between them was 94%. Phylogenetic trees based on 16S rRNA gene sequences revealed that strains J-MR2-Y(T) and J-MR2-O clustered consistently with the type strains of Loktanella soesokkakensis, L. hongkongensis and L. cinnabarina, with which it exhibited 97.83-99.06% sequence similarity. Sequence similarities to the type strains of the other recognized species of the genus Loktanella were 94.01-96.26%. Both strains contained Q-10 as the predominant ubiquinone, C(18 : 1)ω7c as the major fatty acid and phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid and one unidentified aminolipid as the major polar lipids. The DNA G+C contents of strains J-MR2-Y(T) and J-MR2-O were 68.1 and 68.4 mol%, respectively, and DNA-DNA relatedness values with the type strains of L. soesokkakensis, L. hongkongensis and L. cinnabarina were 19-37%. Differential phenotypic properties, together with their phylogenetic and genetic distinctiveness, revealed that the two novel strains are separated from other species of the genus Loktanella. On the basis of the data presented, strains J-MR2-Y(T) and J-MR2-O are proposed to represent a novel species of the genus Loktanella, for which the name Loktanella variabilis sp. nov. is proposed. The type strain is J-MR2-Y(T) ( = KCTC 42074(T) = CECT 8572(T)).

Boseongicola aestuarii gen. nov., sp. nov., isolated from a tidal flat sediment.

A Gram-stain-negative, aerobic, non-motile and pleomorphic (coccoid, ovoid or rod-shaped) bacterial strain, BS-W15(T), isolated from a tidal flat sediment at Boseong in South Korea, was characterized taxonomically. Strain BS-W15(T) grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of approximately 2.0% (w/v) NaCl. Neighbour-joining and maximum-likelihood phylogenetic trees, based on 16S rRNA gene sequences, revealed that strain BS-W15(T) joined the cluster comprising the type strains of Profundibacterium mesophilum, Hwanghaeicola aestuarii, M. pelagius and M. salinus, showing 93.5-96.4% sequence similarities. Strain BS-W15(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c as the predominant fatty acid. The polar lipid profile of strain BS-W15(T) contained phosphatidylcholine and phosphatidylglycerol as major components, differentiating it from those of the type strains of P. mesophilum, H. aestuarii, M. pelagius and M. salinus. The DNA G+C content of strain BS-W15(T) was 58.7 mol%. The differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, demonstrate that strain BS-W15(T) is distinct from type strains of P. mesophilum, H. aestuarii, M. pelagius and M. salinus. On the basis of the data presented, strain BS-W15(T) is considered to represent a novel genus and species, for which the name Boseongicola aestuarii gen. nov., sp. nov. is proposed. The type strain is BS-W15(T) ( = KCTC 32576(T) = CECT 8489(T)).

Mesoflavibacter sabulilitoris sp. nov., isolated from seashore sand.

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated, gliding and rod-shaped bacterial strain, designated GJMS-9(T), was isolated from seashore sand collected at Geoje island in the South Sea, South Korea. Strain GJMS-9(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GJMS-9(T) clustered with the type strain of Mesoflavibacter zeaxanthinifaciens, showing the highest sequence similarity of 99.1%. Strain GJMS-9(T) exhibited 16S rRNA gene sequence similarity of 96.5% to the type strain of Mesoflavibacter aestuarii and of less than 96.1% to the type strains of other recognized species. Strain GJMS-9(T) contained MK-6 as the only menaquinone and iso-C(15:1) G, iso-C(16:0) 3-OH, iso-C(15:0) and iso-C(17:0) 3-OH as the major fatty acids. The polar lipid profile of strain GJMS-9(T) containing phosphatidylethanolamine, one unidentified lipid and one unidentified glycolipid as major components was similar to that of the type strain of M. zeaxanthinifaciens. The DNA G+C content of strain GJMS-9(T) was 32.2 mol% and its DNA-DNA relatedness with M. zeaxanthinifaciens DSM 18436(T) was 38 ± 6.1%. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain GJMS-9(T) is separated from other species of the genus Mesoflavibacter. On the basis of the data presented, strain GJMS-9(T) is considered to represent a novel species of the genus Mesoflavibacter, for which the name Mesoflavibacter sabulilitoris sp. nov. is proposed. The type strain is GJMS-9(T) ( =KCTC 42117(T) =CECT 8597(T)).

Thalassotalea ponticola sp. nov., isolated from seawater, reclassification of Thalassomonas fusca as Thalassotalea fusca comb. nov. and emended description of the genus Thalassotalea.

A Gram-stain-negative, aerobic, non-flagellated and rod-shaped or ovoid bacterial strain, designated GJSW-36(T), was isolated from seawater at Geoje island in the South Sea, South Korea. Strain GJSW-36(T) grew optimally at pH 7.0-8.0, at 25 °C and in the presence of 2% (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain GJSW-36(T) fell within the clade comprising the type strains of species of the genus Thalassotalea and Thalassomonas fusca. Strain GJSW-36(T) exhibited 16S rRNA gene sequence similarity values of 94.2-96.0% to the type strains of species of the genus Thalassotalea and Thalassomonas fusca and of 93.8-94.5% to the type strains of the other species of the genus Thalassomonas. Strain GJSW-36(T) contained ubiquinone-8 (Q-8) as the predominant ubiquinone and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c), C(1:1)ω8c and C(16:0) as the major fatty acids. The major polar lipids of strain GJSW-36(T) were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain GJSW-36(T) was 45.1 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain GJSW-36(T) is separated from species of the genus Thalassotalea and Thalassomonas fusca. On the basis of the data presented, strain GJSW-36(T) is considered to represent a novel species of the genus Thalassotalea, for which the name Thalassotalea ponticola sp. nov. is proposed. The type strain is GJSW-36(T) ( =KCTC 42155(T) =CECT 8656(T)). From this study, it is also proposed that Thalassomonas fusca should be reclassified as a member of the genus Thalassotalea and the description of the genus Thalassotalea is emended.

Ascidiaceihabitans donghaensis gen. nov., sp. nov., isolated from the golden sea squirt Halocynthia aurantium.

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated RSS1-M3(T), was isolated from a golden sea squirt (Halocynthia aurantium) collected from the East Sea, South Korea. Strain RSS1-M3(T) grew optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0 % (w/v) NaCl. Strain RSS1-M3(T) exhibited the highest 16S rRNA gene sequence similarity (96.55 %) to the type strain of Pelagicola litoralis. Neighbour-joining and maximum-likelihood phylogenetic trees based on 16S rRNA gene sequences revealed that strain RSS1-M3(T) clustered with the type strains, or proposed type strains, of Planktotalea frisia, Pacificibacter maritimus, Roseovarius marinus and Halocynthiibacter namhaensis, showing sequence similarity of 94.88-96.32 %. Strain RSS1-M3(T) contained Q-10 as the predominant ubiquinone and C18 : 1ω7c and C16 : 0 as the major fatty acids. The polar lipid profile of strain RSS1-M3(T), containing phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified aminolipid and one unidentified lipid as major components, could be distinguished from those of the phylogenetically related genera. The DNA G+C content of strain RSS1-M3(T) was 55.8 mol%. On the basis of the phylogenetic, chemotaxonomic and phenotypic properties, strain RSS1-M3(T) is considered to represent a novel species of a new genus within the class Alphaproteobacteria, for which the name Ascidiaceihabitans donghaensis gen. nov., sp. nov. is proposed. The type strain is RSS1-M3(T) ( = KCTC 42118(T) = CECT 8599(T)).

Algoriphagus boseongensis sp. nov., a member of the family Cyclobacteriaceae isolated from a tidal flat.

A Gram-stain negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterial strain, BS-R1(T), was isolated from a tidal flat at Boseong, South Korea. Strain BS-R1(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BS-R1(T) belongs to the genus Algoriphagus, clustering consistently with the type strain of Algoriphagus mannitolivorans, with which it exhibited 98.4 % sequence similarity. Sequence similarities between strain BS-R1(T) and the type strains of the other Algoriphagus species were between 92.7 and 97.0 %. Strain BS-R1(T) was found to contain MK-7 as the predominant menaquinone and iso-C15:0, iso-C17:0 3-OH and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) as the major fatty acids. The major polar lipids of strain BS-R1(T) were identified as phosphatidylcholine, phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was determined to be 42.3 mol% and its mean DNA-DNA relatedness values with A. mannitolivorans KACC 11349(T) was 17 ± 5 %. The phylogenetic and genetic distinctiveness and differential phenotypic properties demonstrated that strain BS-R1(T) is distinguishable from the other Algoriphagus species as well as A. mannitolivorans. On the basis of the data presented, strain BS-R1(T) is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus boseongensis sp. nov. is proposed. The type strain is BS-R1(T) (=KCTC 32580(T) = CECT 8446(T)).

Roseovarius gaetbuli sp. nov., a novel alphaproteobacterium isolated from a tidal flat sediment.

A Gram-negative, aerobic, non-motile and ovoid or rod-shaped bacterial strain, designated YM-20(T), was isolated from a tidal flat of the Yellow Sea in South Korea. Strain YM-20(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain YM-20(T) clustered consistently with the type strains of 'Roseovarius marisflavi' and Roseovarius lutimaris, with which it exhibited 16S rRNA gene sequence similarities of 99.86 and 98.71 %, respectively. Strain YM-20(T) was found to contain Q-10 as the predominant ubiquinone and C18:1 ω7c as the predominant fatty acid. The major polar lipids of strain YM-20(T) were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain YM-20(T) was determined to be 60.9 mol% and its DNA-DNA relatedness values with the type strains of 'R. marisflavi', R. lutimaris and Pelagicola litorisediminis were 53 ± 7.1, 22 ± 5.5 and 13 ± 4.7 %, respectively. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain YM-20(T) is separated from 'R. marisflavi', R. lutimaris, the other Roseovarius species and P. litorisediminis. On the basis of the data presented, strain YM-20(T) is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius gaetbuli sp. nov. is proposed. The type strain is YM-20(T) (= KCTC 32428(T) = CECT 8370(T)).

Pseudohongiella acticola sp. nov., a novel gammaproteobacterium isolated from seawater, and emended description of the genus Pseudohongiella.

A Gram-negative, aerobic, non-spore-forming and rod-shaped or ovoid bacterial strain able to move by a single polar flagellum, designated GBSW-5(T), was isolated from seawater in the East Sea, South Korea. Strain GBSW-5(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GBSW-5(T) clustered with the type strain of Pseudohongiella spirulinae. Strain GBSW-5(T) exhibited the highest 16S rRNA gene sequence similarity value (96.4%) to the type strain of P. spirulinae, and of less than 91.0% sequence similarities to the type strains of other recognized species. Strain GBSW-5(T) was found to contain Q-8 as the predominant ubiquinone and C(18:1)ω7c and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c) as the major fatty acids. The major polar lipids of strain GBSW-5(T) were identified as phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The DNA G+C content of strain GBSW-5(T) was determined to be 59.1 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain GBSW-5(T) is separated from P. spirulinae. On the basis of the data presented, strain GBSW-5(T) is considered to represent a novel species of the genus Pseudohongiella, for which the name Pseudohongiella acticola sp. nov. is proposed. The type strain is GBSW-5(T) (= KCTC 42131(T) = CECT 8627(T)). An emended description of the genus Pseudohongiella is also given.

Mariniflexile ostreae sp. nov., a member of the family Flavobacteriaceae isolated from an oyster.

A Gram-negative, aerobic, non-flagellated and rod-shaped bacterial strain able to move by gliding, designated TYO-10(T), was isolated from an oyster collected at Tongyoung on the South Sea, South Korea. Strain TYO-10(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain TYO-10(T) joined the cluster comprising the type strains of Mariniflexile species. Strain TYO-10(T) exhibited 16S rRNA gene sequence similarity values of 95.4-95.7 % to the type strains of Mariniflexile species and of less than 95.4 % to the type strains of other recognized species. Strain TYO-10(T) was found to contain MK-6 as the predominant menaquinone and iso-C15:0, iso-C15:1 G, anteiso-C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH as the major fatty acids. The major polar lipids of strain TYO-10(T) were identified as phosphatidylethanolamine and an unidentified lipid, which is similar to those of the type strains of Mariniflexile species, but different from those of other phylogenetically related species. The DNA G+C content of strain TYO-10(T) was determined to be 35.9 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain TYO-10(T) is separated from other Mariniflexile species. On the basis of the data presented, strain TYO-10(T) is considered to represent a novel species of the genus Mariniflexile, for which the name Mariniflexile ostreae sp. nov. is proposed. The type strain is TYO-10(T) (= KCTC 42113(T) = CECT 8622(T)).

Loktanella aestuariicola sp. nov., an alphaproteobacterium isolated from a tidal flat.

A Gram-stain negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, J-TF4(T), which was isolated from a tidal flat in the South Sea of South Korea, was characterized taxonomically. Strain J-TF4(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0-3.0% (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain J-TF4(T) fell within the clade comprising the type strains of Loktanella species, clustering with the type strains of Loktanella cinnabarina, Loktanella hongkongensis, Loktanella soesokkakensis, Loktanella pyoseonensis and 'Loktanella variabilis' showing sequence similarity values of 97.2-98.4%. The strain exhibited 16S rRNA gene sequence similarity values of 94.1-95.4% to the type strains of the other Loktanella species. Strain J-TF4(T) was found to have Q-10 as the predominant ubiquinone and C(18:1) ω7c as the major fatty acid. The major polar lipids of strain J-TF4(T) were identified as phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and an unidentified aminolipid. The DNA G+C content of strain J-TF4(T) was determined to be 67.9 mol% and its mean DNA-DNA relatedness values with the type strains of five phylogenetically related Loktanella species were 17.7-23.3%. Differential phenotypic properties, together with the phylogenetic and genetic data, demonstrate that strain J-TF4(T) is separated from other Loktanella species. On the basis of the data presented, strain J-TF4(T) is considered to represent a novel species of the genus Loktanella, for which the name Loktanella aestuariicola sp. nov. is proposed. The type strain is J-TF4(T) (=KCTC 42135(T)=NBRC 110408(T)).

Granulosicoccus undariae sp. nov., a member of the family Granulosicoccaceae isolated from a brown algae reservoir and emended description of the genus Granulosicoccus.

A Gram-stain-negative, aerobic, non-flagellated and coccoid bacterial strain, W-BA3(T), which was isolated from a brown algae reservoir in Wando of South Korea, was characterized taxonomically. Strain W-BA3(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain W-BA3(T) clustered with the type strains of Granulosicoccus antarcticus and Granulosicoccus coccoides, with which it exhibited sequence similarity values of 98.4-99.3 %. Sequence similarity values of strain W-BA3(T) to the type strains of the other recognized species were less than 90.2 %. Strain W-BA3(T) was found to contain Q-8 as the predominant ubiquinone and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C18:1 ω7c and C16:0 as the major fatty acids. The major polar lipids of strain W-BA3(T), which were identified as phosphatidylethanolamine and phosphatidylglycerol, were similar to those of the type strains of G. antarcticus and G. coccoides. The DNA G+C content of strain W-BA3(T) was 56.0 mol % and its mean DNA-DNA relatedness values with the type strains of G. coccoides and G. antarcticus were 27 and 17 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain W-BA3(T) is separated from the two Granulosicoccus species. On the basis of the data presented, strain W-BA3(T) is considered to represent a novel species of the genus Granulosicoccus, for which the name Granulosicoccus undariae sp. nov. is proposed. The type strain is W-BA3(T) (=KCTC 42134(T) = NBRC 110411(T)). An emended description of the genus Granulosicoccus is also proposed.

Thalassobius aquaeponti sp. nov., an alphaproteobacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, GJSW-22(T), which was isolated from seawater at Geoje island in South Korea, was characterized taxonomically. Strain GJSW-22(T) was observed to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22(T) grouped with the type strains of Thalassobius species, forming a stable cluster with the type strain of Thalassobius aestuarii (bootstrap value of 83.2 %). Strain GJSW-22(T) exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of T. aestuarii. It exhibited 16S rRNA gene sequence similarity values of 95.6-96.1 % to the type strains of the other Thalassobius species. Strain GJSW-22(T) was found to contain Q-10 as the predominant ubiquinone and C18:1 ω7c and 11-methyl C18:1 ω7c as the major fatty acids. The major polar lipids of strain GJSW-22(T) were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of strain GJSW-22(T) is 60.3 mol % and its mean DNA-DNA relatedness value with the type strain of T. aestuarii was 23 %. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, confirmed that strain GJSW-22(T) is distinct from other Thalassobius species. On the basis of the data presented, strain GJSW-22(T) is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed. The type strain is GJSW-22(T) (=KCTC 42115(T) = NBRC 110378(T)).

Invasiveness of Upper Tract Urothelial Carcinoma: Clinical Significance and Integrative Diagnostic Strategy.

PURPOSE: In this study, we aimed to determine the clinicopathologic, radiologic, and molecular significance of the tumor invasiveness to further stratify the patients with high-grade (HG) upper tract urothelial carcinoma (UTUC) who can be treated less aggressively. MATERIALS AND METHODS: Clinicopathologic and radiologic characteristics of 166 surgically resected HG UTUC (48 noninvasive, and 118 invasive) cases were evaluated. Six noninvasive UTUC cases with intratumoral tumor grade heterogeneity were selected for whole-exome sequencing (WES) to understand the underlying molecular pathophysiology. Barcode-tagging sequencing was done for validation of the target genes from WES data. RESULTS: Patients with noninvasive UTUC showed no cancer-specific death with better cancer-specific survival (p < 0.001) and recurrence-free survival (p < 0.001) compared to the patients with invasive UTUC. Compared to the invasive UTUC, noninvasive UTUC was correlated to a low grade (LG) on the preoperative abdominal computed tomography (CT) grading system (p < 0.001), histologic intratumoral tumor grade heterogeneity (p=0.018), discrepancy in preoperative urine cytology diagnosis (p=0.018), and absence of urothelial carcinoma in situ (p < 0.001). WES of the heterogeneous components showed mutually shared HRAS and FGFR3 mutations shared between the HG and LG components. HRAS mutation was associated with the lower grade on preoperative abdominal CT and intratumoral tumor grade heterogeneity (p=0.045 and p < 0.001, respectively), whereas FGFR3 mutation was correlated to the absence of carcinoma in situ (p < 0.001). CONCLUSION: According to our comprehensive analysis, HG noninvasive UTUC can be preoperatively suspected based on distinct preoperative radiologic, cytologic, histologic, and molecular features. Noninvasive HG UTUC shows excellent prognosis and thus should be treated less aggressively.

Artificial intelligence algorithm for neoplastic cell percentage estimation and its application to copy number variation in urinary tract cancer.

Background: Bladder cancer is characterized by frequent mutations, which provide potential therapeutic targets for most patients. The effectiveness of emerging personalized therapies depends on an accurate molecular diagnosis, for which the accurate estimation of the neoplastic cell percentage (NCP) is a crucial initial step. However, the established method for determining the NCP, manual counting by a pathologist, is time-consuming and not easily executable. Methods: To address this, artificial intelligence (AI) models were developed to estimate the NCP using nine convolutional neural networks and the scanned images of 39 cases of urinary tract cancer. The performance of the AI models was compared to that of six pathologists for 119 cases in the validation cohort. The ground truth value was obtained through multiplexed immunofluorescence. The AI model was then applied to 41 cases in the application cohort that underwent next-generation sequencing testing, and its impact on the copy number variation (CNV) was analyzed. Results: Each AI model demonstrated high reliability, with intraclass correlation coefficients (ICCs) ranging from 0.82 to 0.88. These values were comparable or better to those of pathologists, whose ICCs ranged from 0.78 to 0.91 in urothelial carcinoma cases, both with and without divergent differentiation/ subtypes. After applying AI-driven NCP, 190 CNV (24.2%) were reclassified with 66 (8.4%) and 78 (9.9%) moved to amplification and loss, respectively, from neutral/minor CNV. The neutral/minor CNV proportion decreased by 6%. Conclusions: These results suggest that AI models could assist human pathologists in repetitive and cumbersome NCP calculations.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

PURPOSE: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. MATERIALS AND METHODS: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. RESULTS: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. CONCLUSIONS: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

PD-L1 Upregulation by the mTOR Pathway in VEGFR-TKI-Resistant Metastatic Clear Cell Renal Cell Carcinoma.

PURPOSE: Tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) signaling pathways have been used for metastatic clear cell renal cell carcinoma (mCCRCC), but resistance to the drug develops in most patients. We aimed to explore the underlying mechanism of the TKI resistance with regard to programmed death-ligand 1 (PD-L1) and to investigate signaling pathway associated with the resistant mechanism. MATERIALS AND METHODS: To determine the mechanism of resistance, 10 mCCRCC patients from whom tumor tissues were harvested at both the pretreatment and the TKI-resistant post-treatment period were included as the discovery cohort, and their global gene expression profiles were compared. A TKI-resistant renal cancer cell line was established by long-term treatment with sunitinib. RESULTS: Among differentially expressed genes in the discovery cohort, increased PD-L1 expression in post-treatment tissues was noted in four patients. Pathway analysis showed that PD-L1 expression was positively correlated with the mammalian target of rapamycin (mTOR) signaling pathway. The TKI-resistant renal cancer cells showed increased expression of PD-L1 and mTOR signaling proteins and demonstrated aggressive tumoral behaviour. Treatment with mTOR inhibitors down-regulated PD-L1 expression and suppressed aggressive tumoral behaviour, which was reversed with stimulation of the mTOR pathway. CONCLUSION: These results showed that PD-L1 expression may be increased in a subset of VEGFR-TKI-resistant mCCRCC patients via the mTOR pathway.