FGFR3 Mutations in Urothelial Carcinoma: A Single-Center Study Using Next-Generation Sequencing.

Background: Mutations of fibroblast growth factor receptor 3 (FGFR3) are associated with urothelial carcinoma (UC) oncogenesis and are considered an important therapeutic target. Therefore, we evaluated the FGFR3 mutation rate and its clinical significance in urothelial carcinoma (UC) using next-generation sequencing. Methods: A total of 123 patients with UC who were treated at Chonnam National University Hospital (Gwang-ju, Korea) from January 2018 to December 2020 were enrolled. We performed NGS using the Oncomine panel with tumor specimens and blood samples corresponding to each specimen. We analyzed the FGFR3 mutation results according to the type of UC and the effects on early recurrence and progression. Results: The mean age of the patients was 71.39 ± 9.33 years, and 103 patients (83.7%) were male. Overall, the FGFR3 mutation rate was 30.1% (37 patients). The FGFR3 mutation rate was the highest in the non-muscle-invasive bladder cancer (NMIBC) group (45.1%), followed by the muscle-invasive bladder cancer (22.7%) and upper tract UC (UTUC) (14.3%) groups. Patients with FGFR3 mutations had a significantly lower disease stage (p = 0.019) but a high-risk of NMIBC (p < 0.001). Conclusions: Our results revealed that FGFR3 mutations were more prevalent in patients with NMIBC and lower stage UC and associated with a high-risk of NMIBC. Large multicenter studies are needed to clarify the clinical significance of FGFR3 mutations in UC.

Somatic Mutation of the Non-Muscle-Invasive Bladder Cancer Associated with Early Recurrence.

Next-generation sequencing (NGS) is widely used in muscle-invasive bladder cancer but has limited use in non-muscle-invasive bladder cancer (NMIBC) due to significant heterogeneity and high cancer-specific survival. Therefore, we evaluated the genomic information of NMIBC and identified molecular alterations associated with tumour recurrence. A total of 43 patients with NMIBC who underwent transurethral resection of the bladder were enrolled. We performed NGS using an Oncomine panel of tumour specimens and blood samples corresponding to each specimen. The somatic mutation results were analysed by pairwise comparison and logistic regression according to the recurrence of bladder tumours within 1 year. The median incidence of genetic variations in 43 tumour samples was 56 variations per sample, and a high tumour mutation burden (TMB) was associated with tumour recurrence (median variation 33 vs. 64, p = 0.023). The most mutated gene was adipose tissue macrophages (ATM) (79%), followed by neurofibromatosis-1 (NF1) (79%), and neurogenic locus notch homolog protein 1 (NOTCH1) (79%). In multivariable analysis, mutation of epidermal growth factor receptor (EGFR) (odds ratio [OR], 9.95; 95% confidence interval [CI], 1.40-70.96; p = 0.022) and telomerase reverse transcriptase (TERT) (OR, 7.92; 95% CI, 1.22-51.51; p = 0.030) were significant factors associated with the recurrence of bladder tumour within 1 year. Our results revealed that high TMB, EGFR mutation, and TERT mutation had a significant association with tumour recurrence in NMIBC. In addition, somatic mutations in EGFR and TERT could be useful prognostic biomarkers in NMIBC.

Naturally occurring hepatitis B virus X deletions and insertions among Korean chronic patients.

Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection.

Development and application of multiprobe real-time PCR method targeting the hsp65 gene for differentiation of Mycobacterium species from isolates and sputum specimens.

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.

First case of disseminated Mycobacterium bolletii infection in a young adult patient.

Mycobacterium bolletii is a rapidly growing nontuberculous mycobacterium first characterized in 2006. Here, we report a case of disseminated infection caused by M. bolletii in a young adult patient. To our knowledge, this is the first case of disseminated M. bolletii infection in an otherwise healthy young adult.

Ankle Fusion Combined With Calcaneal Sliding Osteotomy for Severe Arthritic Ball and Socket Ankle Deformity.

BACKGROUND: Although a ball and socket ankle deformity is usually congenital and asymptomatic, abnormal inversion and eversion mobility can result in recurrent ankle sprain and osteoarthritis. This retrospective study was performed to evaluate the clinical and radiologic outcomes of ankle fusion combined with calcaneal sliding osteotomy for severe arthritic ball and socket ankle deformity. METHODS: Fourteen patients with severe arthritic ball and socket ankle deformity were followed for more than 3 years after operation. The clinical evaluation consisted of American Orthopaedic Foot & Ankle Society (AOFAS) score, Foot and Ankle Ability Measure (FAAM), visual analog scale (VAS) for pain, and subjective satisfaction score. The period to fusion and union of osteotomy, the change of hindfoot alignment angle, and complications were evaluated radiologically. RESULTS: AOFAS and FAAM scores were significantly improved from an average of 37.4 and 34.5 points to 74.6 and 78.5 points, respectively. VAS for pain with walking over 20 minutes was significantly improved from an average of 8.4 points to 1.9 points. The average satisfaction score of patients was 88.9 points. The difference in heel alignment angle (compared to contralateral side) was significantly improved from an average of 34.8 to 5.4 degrees. There were 2 cases of progressive arthritis in an adjacent joint and 1 case of failed fusion. CONCLUSIONS: Ankle fusion combined with calcaneal sliding osteotomy can be an effective operative option for ball and socket ankle deformity with advanced arthritis. In spite of increased complication rate, reliable pain relief, and restoration of gait ability through correcting hindfoot malalignment could improve the quality of life. LEVEL OF EVIDENCE: Level IV, retrospective case series.

The development of a novel Mycobacterium-Escherichia coli shuttle vector system using pMyong2, a linear plasmid from Mycobacterium yongonense DSM 45126T.

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.

Whole-Genome Sequence of Mycobacterium intracellulare Clinical Strain MOTT-H4Y, Belonging to INT5 Genotype.

Here, we report the draft genome sequence of the Mycobacterium intracellulare clinical strain MOTT-H4Y, grouped previously into the INT5 genotype of the 5 genotypes of M. intracellulare.

Molecular characterization of Mycobacterium intracellulare-related strains based on the sequence analysis of hsp65, internal transcribed spacer and 16S rRNA genes.

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex.

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB, confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05-2522 (= DSM 45127 = KCTC 19556).