Relationships between Secreted Aspartyl Proteinase 2 and General Control Nonderepressible 4 gene in the Candida albicans resistant to itraconazole under planktonic and biofilm conditions.

This study aimed to explore the roles of SAP2 and GCN4 in itraconazole (ITR) resistance of C. albicans under different conditions, and their correlations. A total of 20 clinical strains of C. albicans, including 10 ITR resistant strains and 10 sensitive strains, were used. Then, SAP2 sequencing and GCN4 sequencing were performed, and the biofilm formation ability of different C. albicans strains was determined. Finally, real-time quantitative PCR was used to measure the expression of SAP2 and GCN4 in C. albicans under planktonic and biofilm conditions, as well as their correlation was also analyzed. No missense mutations and three synonymous mutation sites, including T276A, G543A, and A675C, were found in SAP2 sequencing. GCN4 sequencing showed one missense mutation site (A106T (T36S)) and six synonymous mutation sites (A147C, C426T, T513C, T576A, G624A and C732T). The biofilm formation ability of drug-resistant C. albicans strains was significantly higher than that of sensitive strains (P < 0.05). Additionally, SAP2 and GCN4 were up-regulated in the ITR-resistant strains, and were both significantly higher in C. albicans under biofilm condition. The mRNA expression levels of SAP2 and GCN4 had significantly positive correlation. The higher expression levels of SAP2 and GCN4 were observed in the ITR-resistant strains of C. albicans under planktonic and biofilm conditions, as well as there was a positive correlation between SAP2 and GCN4 mRNA expression.

[Analysis of KIF1A gene variant in a Chinese pedigree affected with Spastic paraplegia type 30].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with hereditary spastic paraplegia type 30 (HSP30). METHODS: A proband presented at the Second Hospital of Shanxi Medical University in August 2021 was selected as the study subject. The proband was subjected to whole exome sequencing, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The proband was found to have harbored a heterozygous c.110T>C variant in exon 3 of the KIF1A gene, which can cause substitution of isoleucine by threonine at position 37 (p.I37T) and alter the function of its protein product. The same variant was not found in his parents, elder brother and elder sister, suggesting that it has a de novo origin. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic (PM2_Supporting+PP3+PS2). CONCLUSION: The c.110T>C variant of the KIF1A gene probably underlay the HSP30 in the proband. Above finding has enable genetic counseling for this family.

Correlation between SAP2 and CAP1 in clinical strains of Candida albicans at planktonic and biofilm states.

This study aimed to explore the influences of SAP2 and CAP1 on itraconazole (ITR) resistance of Candida albicans at different states. A total of 10 ITR-resistant strains and 10 ITR-sensitive strains were used for SAP2 sequencing and CAP1 sequencing. SAP2 sequencing showed no missense mutation, and three synonymous mutations. CAP1 gene sequencing identified two missense mutations M140I (8) and K191Q (4), and 14 synonymous mutations G201A (1), A246C (5), C282T (6), G288A (6), C321T (7), A399C (16), C432T (16), C465T (11), G552A (16), G669T (1), G672A (1), G681T (2), T783C (1), and T819A (2). The biofilm formation capacity of resistant C. albicans strains, including the CAP1∆/∆ strain, was stronger. Afterward, real-time quantitative PCR was used to analyze the expression of SAP2 and CAP1. Compared with the sensitive strains, SAP2 and CAP1 expressions were both significantly upregulated in resistant strains at planktonic and biofilm states (P < 0.05). Compared with the strains at planktonic state, SAP2 was significantly upregulated, while CAP1 was significantly downregulated at biofilm states (P < 0.05). Additionally, SAP2 expression in the CAP1 knocked down strain of C. albicans was significantly upregulated, and SAP2 expression was evidently downregulated in the CAP1∆/∆ strain at biofilm states compared with that at planktonic states (P < 0.05). Loss of CAP1 can increase SAP2 level and may influence the biofilm formation of C. albicans, thus increasing ITR resistance ofC. albicans.

Case Report: A Missense Mutation in Dyskeratosis Congenita 1 Leads to a Benign Form of Dyskeratosis Congenita Syndrome With the Mucocutaneous Triad.

Background: Dyskeratosis congenita (DC) is a rare inheritable disorder characterized by bone marrow failure and mucocutaneous triad (reticular skin pigmentation, nail dystrophy, and oral leukoplakia). Dyskeratosis congenita 1 (DKC1) is responsible for 4.6% of the DC with an X-linked inheritance pattern. Almost 70 DKC1 variations causing DC have been reported in the Human Gene Mutation Database. Results: Here we described a 14-year-old boy in a Chinese family with a phenotype of abnormal skin pigmentation on the neck, oral leukoplakia, and nail dysplasia in his hands and feet. Genetic analysis and sequencing revealed hemizygosity for a recurrent missense mutation c.1156G > A (p.Ala386Thr) in DKC1 gene. The heterozygous mutation (c.1156G > A) from his mother and wild-type sequence from his father were obtained in the same site of DKC1. This mutation was determined as disease causing based on silico software, but the pathological phenotypes of the proband were milder than previously reported at this position (HGMDCM060959). Homology modeling revealed that the altered amino acid was located near the PUA domain, which might affect the affinity for RNA binding. Conclusion: This DKC1 mutation (c.1156G > A, p.Ala386Thr) was first reported in a Chinese family with mucocutaneous triad phenotype. Our study reveals the pathogenesis of DKC1 c.1156G > A mutation to DC with a benign phenotype, which expands the disease variation database, the understanding of genotype-phenotype correlations, and facilitates the clinical diagnosis of DC in China.

Genetic and functional analyses detect an EXT1 splicing pathogenic variant in a Chinese hereditary multiple exostosis (HME) family.

BACKGROUND: Hereditary multiple exostosis (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple cartilage-covered tumors on the external surfaces of bones (osteochondromas). Most of HME cases result from heterozygous loss-of-function mutations in EXT1 or EXT2 gene. METHODS: Clinical examination was performed to diagnose the patients: Whole exome sequencing (WES) was used to identify pathogenic mutations in the proband, which is confirmed by Sanger sequencing and co-segregation analysis: qRT-PCR was performed to identify the mRNA expression level of EXT1 in patient peripheral blood samples: minigene splicing assay was performed to mimic the splicing process of EXT1 variants in vitro. RESULTS: We evaluated the pathogenicity of EXT1 c.1056 + 1G > T in a Chinese family with HME. The clinical, phenotypic, and genetic characterization of patients in this family were described. The variant was detected by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Sequencing of the RT-PCR products from the patient's blood sample identified a large deletion (94 nucleotides), which is the whole exome 2 of the EXT1 cDNA. Splicing assay indicated that the mutated minigene produced alternatively spliced transcripts, which cause a frameshift resulting in an early termination of protein expression. CONCLUSIONS: Our study establishes the pathogenesis of the splicing mutation EXT1 c.1056 + 1G > T to HME and provides scientific foundation for accurate diagnosis and precise medical intervention for HME.

[Identification of a novel SOD1 variant in a Chinese patient with amyotrophic lateral sclerosis].

OBJECTIVE: To explore the genetic basis for a Chinese patient with amyotrophic lateral sclerosis (ALS). METHODS: Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Genetic variant was identified by whole exome sequencing. Candidate variant was verified by Sanger sequencing of his parents and healthy controls. RESULTS: The patient was found to harbor a heterozygous c.420C>G (p.Asn140Lys) variant of the SOD1 gene. The same variant was not detected in his parents and 100 healthy controls. The variant has not been included in HGMD, dbSNP and other databases. CONCLUSION: The c.420C>G variant of the SOD1 gene may underlie the ALS in this patient. Above finding has enriched the spectrum of SOD1 gene variants.

[Analysis of PKD2 gene variant and protein localization in a pedigree affected with polycystic kidney disease].

OBJECTIVE: To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease (ADPKD) and verify its impact on the protein function. METHODS: Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization. RESULTS: The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene. CONCLUSION: The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.

A novel splicing pathogenic variant in COL1A1 causing osteogenesis imperfecta (OI) type I in a Chinese family.

BACKGROUND: Osteogenesis imperfecta (OI), a rare autosomal inheritable disorder characterized by bone fragility and skeletal deformity, is caused by pathogenic variants in genes impairing the synthesis and processing of extracellular matrix protein collagen type I. With the use of next-generation sequencing and panels approaches, an increasing number of OI patients can be confirmed and new pathogenic variants can be discovered. This study sought to identify pathogenic gene variants in a Chinese family with OI I. METHODS: Whole-exome sequencing was used to identify pathogenic variants in the proband, which is confirmed by Sanger sequencing and cosegregation analysis; MES, HSF, and Spliceman were used to analyze this splicing variant；qRT-PCR was performed to identify the mRNA expression level of COL1A1 in patient peripheral blood samples; Minigene splicing assay was performed to mimic the splicing process of COL1A1 variants in vitro; Analysis of evolutionary conservation of amino acid residues and structure prediction of the mutant protein. RESULTS: A novel splicing pathogenic variant (c.3814+1G>T) was identified in this OI family by using whole-exome sequencing, Sanger sequencing, and cosegregation analysis. Sequencing of RT-PCR products from the COL1A1 minigene variant reveals a 132-nucleotide (nt) insertion exists at the junction between exons 48 and exon 49 of the COL1A1 cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which may cause a frameshift resulting in early termination of protein expression. The molecular analysis suggested that the altered amino acid is located at the C-terminus of type I procollagen. CONCLUSION: Our study reveals the pathogenesis of a novel COL1A1 splicing pathogenic variant c.3814+1G>T in a Chinese family with OI I.

Preliminary evaluation and discussion of the safety of left innominate vein resection.

BACKGROUND: To evaluate the safety of resection of anterior mediastinal lesions involving the left innominate vein (LIV) and analyze the risk factors affecting LIV resection safety. METHODS: Patients who underwent anterior mediastinal lesion and LIV resection from January 2010 to December 2018 in the Department of Thoracic Surgery of Tangdu Hospital, Air Force Medical University, were followed up, and preoperative, intraoperative and postoperative factors were analyzed. RESULTS: Forty-eight patients who underwent anterior mediastinal lesion and LIV resection from January 2010 to December 2018, except for 2 who died of lung infection-induced respiratory failure, were followed up, with an average follow-up time of 32 months (range, 6-72 months). Postoperative: in 31 cases (67.39%), patients did not manifest LIV resection-associated complications; in 15 cases (32.61%), patients manifested mild LIV resection-associated complications; no patient manifested severe LIV resection-associated complications. The average operation time, average blood loss and average hospitalization time were 155.17 min, 324.13 mL and 11.83 days, respectively. Univariate analysis showed that the degree of LIV invasion and surgical approach were risk factors for predicting LIV resection safety. CONCLUSIONS: For anterior mediastinal lesions involving the LIV, LIV resection is a simple, safe and effective surgical procedure.

Identification of a novel compound heterozygous IDUA mutation underlies Mucopolysaccharidoses type I in a Chinese pedigree.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is a rare autosomal storage disorder resulting from the defective alpha-L-iduronidase (encoded by IDUA) enzyme activity and accumulation of glycosaminoglycans (GAGs) in lysosomes. So far, more than 100 IDUA causative mutations have been identified leading to three MPS I phenotypic subtypes: Hurler syndrome (severe form), Hurler/Scheie syndrome (intermediate form), and Scheie syndrome (mild form). METHODS: Whole-exome sequencing (WES) was performed to identify the underlying genetic mutations. To verify the identified variations, Sanger sequencing was performed for all available family members following PCR amplification. The impact on IDUA protein was analyzed by sequential analysis and homology modeling. RESULTS: A novel IDUA heterozygous single base insertion (c.1815dupT, p.V606Cfs51* ) and a known missence mutation (c.T1037G, p.L346R) were detected in our patient diagnosed as congenital heart disease with heart valve abnormalities. The novel frameshift mutation results in a complete loss of 48 amino acids in the Ig-like domain and causes the formation of a putative protein product which might affect the IDUA enzyme activity. CONCLUSIONS: A novel compound heterozygous IDUA mutation (c.1815dupT, p.V606Cfs51* ) was found in a Chinese MPS I family. The identification of the mutation facilitated accurate genetic counseling and precise medical intervention for MPS I in China.

Functional evaluation of a novel GLA causative mutation in Fabry disease.

BACKGROUND: Fabry disease (FD), a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide in a variety of cell types. More and more disease-causing mutations in GLA have been identified in FD due to the advancement of molecular diagnostic tools. We found a novel mutation in a Chinese family with predominant Fabry's disease nephropathy. METHODS: All coding regions and exon-intron splice junctions of the GLA gene were sequenced to find sequence variations. We evaluated the impact on the GLA protein by analysis of the GLA mRNA, by sequential analysis and homology modeling, and by site-directed mutagenesis and in vitro expression studies. RESULTS: We identified a novel heterozygous missense mutation c.280T>C in our patient with variable phenotypic presentations of renal involvement. The novel GLA variant results in low expression of GLA mRNAs, impaired or loss of the disulfate bridge structure of wild-type GLA, reduced GLA activity and defected nuclear shape in the GFP-GLA-MT transfected HEK293T cells. CONCLUSION: A novel GLA missense mutation, c.280T>C (Cys94Arg), was found in a Chinese family with predominant renal manifestations of FD. Our study reveals the pathogenesis of c.280T>C mutation to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

[Instability of Mitochondrial DNA D-loop Region Genes in Patients with Leukemia].

OBJECTIVE: To study the instability of mitochondrial DNA（mt DNA） D-loop region genes in patients with Leukemia. METHODS: The HV-1 and HV-2 regions of D-loop region in 24 patients with leukemia were amplificated and sequenced, then their results were compared with revised Cambridge reference sequence (rCRS) and Databank mtDB. The mutation rate was detected by SPSS 22.0 statistics software. RESULTS: The total mutation rate in patients was 95.83% (23/24), the detection showed 82 mutated genes, out of which 47 (57.32%) mutated genes located in HV-1 region, 35 (42.68%) mutated genes in HV-2 region. The comparison showed that the mutation rate in untreated (UT) group and treated (T) group of AML patients was (2.37±0.82)×10-3 and (4.76±2.45)×10-3 respectively(P＜0.01), the mutation rate in PR and CR groups of treated AML patients was (5.10±2.56)×10-3 and (4.51±2.51)×10-3 respectively (P＜0.05), the comparison among M3 group showed that the mutation rates in UT, PR and CR groups were (2.55±0.63)×10-3, (5.37±3.41)×10-3 and (3.71±1.65)×10-3 respectively (P＞0.05). CONCLUSION: The more high mutation rate and many kinds of mutation types exist in D-loop region, suggesting that the genes in D-loop region display the more strong instability, the chemotherapy may aggravate the instability of genes in D-loop region.

A Novel α-Galactosidase A Splicing Mutation Predisposes to Fabry Disease.

Fabry disease (FD) is a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide (Gb3) in a variety of cell types. Here, we report a novel splicing mutation (c.801 + 1G > A) that results in alternative splicing in GLA of a FD patient with variable phenotypic presentations of renal involvement. Sequencing of the RT-PCR products from the patient's blood sample reveals a 36-nucleotide (nt) insertion exists at the junction between exons 5 and 6 of the GLA cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which causes a frameshift resulting in an early termination of protein expression. Immunofluorescence shows puncta in cytoplasm for mutated GLA whereas uniform staining small dots evenly distributed inside cytoplasm for wild type GLA in transfected HeLa cells. The increased senescence and decreased GLA enzyme activity suggest that the abnormalities might be due to the altered localization which further might result from the lack of the C-terminal end of GLA. Our study reveals the pathogenesis of splicing mutation c.801 + 1G > A to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

GJB2 c.235delC variant associated with autosomal recessive nonsyndromic hearing loss and auditory neuropathy spectrum disorder.

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is a genetically heterogeneous neurosensory disorder, usually characterized by congenital or prelingual hearing loss. We report a Han Chinese male, born to consanguineous parents, presenting with nonsyndromic sensorineural hearing loss, whose clinical phenotype was also consistent with auditory neuropathy spectrum disorder (ANSD). After exome sequencing, a gap junction protein beta 2 gene (GJB2) c.235delC variant in the homozygous state was detected in the patient. Both parents were heterozygous for this variant, as documented by Sanger sequencing. The known pathogenic GJB2 c.235delC variant was not detected in 200 healthy controls. It is predicted to be a disease-causing alteration by generating a truncated protein p.(L79Cfs*3), disturbing the appropriate folding and/or oligomerization of connexins and leading to defective gap junction channels. This study shows that the association of homozygosity of the GJB2 c.235delC variant with ARNSHL and ANSD in a patient.

Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression.

Regulation of cancer angiogenesis could be a useful strategy in cancer therapy. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA), and can induce cancer cell proliferation, while lncRNAs, generally are able to act as microRNA (miRNA) sponges. The latter is a type of competitive endogenous RNA (ceRNA) that regulates expression of the targeting miRNAs and protein-coding genes. This study investigated the proliferative role of MALAT1 in human umbilical vein endothelial cells (HUVECs) and the underlying molecular events. The data showed that knockdown of MALAT1 expression using MALAT1 siRNA inhibited HUVEC proliferation and also significantly decreased levels of FOXM1 mRNA and protein in vitro, while knockdown of FOXM1 expression reduced HUVEC proliferation. Annotation of HUVEC microarray data revealed that seven miRNAs, including miR-320a, were upregulated after knockdown of MALAT1 expression in HUVECs. MALAT1 was shown to reciprocally interact with miR-320a, i.e., expression of one negatively regulated levels of the other, whereas knockdown of MALAT1 expression promoted miR-320a levels. Furthermore, miR-320a could directly target and inhibit FOXM1 expression in HUVECs. Knockdown of MALAT1 expression enhanced miR-320a expression but reduced FOXM1 expression resulting in downregulation of HUVEC proliferation. However, such an effect was inhibited by miR-320a depletion. In conclusion, this study demonstrates that miR-320a plays an important role in mediating the effects of MALAT1 on HUVEC proliferation by suppression of FOXM1 expression. Thus, targeting of this gene pathway could be a novel strategy in cancer therapy.

Effect of cauterizing esophageal mucosa in "double-patch" treatment for acquired benign tracheoesophageal fistula/bronchogastric stump fistula.

BACKGROUND: The management of acquired benign tracheoesophageal fistula (TEF) and bronchogastric stump fistula (BGSF) is a challenge. This study aimed to assess the "double-patch" technique with or without esophageal mucosa in treating nonmalignant TEF and BGSF. MATERIALS AND METHODS: We established a dog model with TEF by incising the esophageal and tracheal membranes and suturing them together. The dogs were divided into three groups (n = 12 per group). Groups A and B received a double-patch 7 d later. The esophageal mucosa of the patches was cauterized in the group A dogs, kept intact in group B dogs, and group C dogs did not receive surgical intervention. Tissue healing was measured using hydroxyproline levels. RESULTS: Morphologic and histopathologic changes of the esophagus were assessed by gross observation of the specimens, hematoxylin and eosin staining, tracheal stenosis index, and hydroxyproline levels. On day 56 after surgery, group A showed a tracheal stenosis index comparable with that of group C (0.140 ± 0.009 versus 0.138 ± 0.014, P = 1.00), whereas group B showed a higher stenosis index (0.170 ± 0.007) than group C (P = 0.029). The hydroxyproline levels were higher in group A than in B and C on day 7 (P = 0.029), and this difference was statistically significant on days 14 and 56 (all P < 0.001). CONCLUSIONS: The use of an esophageal "double-patch" technique without mucosa showed faster and more stable recovery than patches with mucosa in the repair of acquired nonmalignant complicated TEF and BGSF.

Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer.

BACKGROUND: The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. METHODS: EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients' survival. RESULTS: A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0.092), but esophageal cancer patients or ESCC patients with low EGFR gene copy number may have longer survival than those with high EGFR gene copy number according to the survival curve trends. CONCLUSIONS: The results indicated that EGFR or K-ras mutation was rare in esophageal cancer, but high EGFR gene copy number is frequent, and correlated with advanced pathologic stage and more number of the metastatic regional lymph nodes, especially in ESCC. In addition, high EGFR gene copy number is likely to have a deleterious effect on prognosis of esophageal cancer patients or ESCC patients, although no statistical significance was reached in the study.

A novel surgical method for acquired non-malignant complicated tracheoesophageal and bronchial-gastric stump fistula: the "double patch" technique.

BACKGROUND: To manage the acquired benign complicated tracheoesophageal fistula (TEF) and bronchial-gastric stump fistula (BGSF) are clinical technical challenge. The purpose of this study is to retrospectively review a surgical "double patch" technique in treating nonmalignant complicated TEF and BGSF, and then clarify the long-term curative effect of the technique. METHODS: Clinical records of 30 patients with non-malignant complicated TEF and BGSF treated by "double patch" technique in Tangdu Hospital between August 2004 and August 2014, were analyzed and summarized retrospectively. RESULTS: Thirty patients (19 males and 11 females) underwent "double patch" surgical repair of acquired benign complicated TEF and BGSF. The median age of the patients was 40.2±21.1 years. The most common causes were the following: TEF [22], BGSF [8]. Post-intubation injury [6], trauma [5], foreign body and stents [10], complications from prior esophageal surgery [8], and caustic ingestion [1]. The follow-up was completed for 24 months in all the patients (100%). The operative mortality was 0% (0/30). Twenty-six patients (86.7%) recovered uneventfully while four patients (13.3%) exhibited some major complications in the perioperative and postoperative periods. One patient (3.3%) developed recurrence of tracheal fistula in situ, two patients (6.7%) showed pneumonia, and one patient (3.3%) developed fistula esophageal anastomosis. All the 30 patients resumed oral intake finally. CONCLUSIONS: The double patch technique is an effective and safe method to repair the acquired non-malignant complicated TEF and BGSF.

High ABCG4 Expression Is Associated with Poor Prognosis in Non-Small-Cell Lung Cancer Patients Treated with Cisplatin-Based Chemotherapy.

ATP-binding cassette (ABC) transporters are associated with poor response to chemotherapy, and confer a poor prognosis in various malignancies. However, the association between the expression of the ABC sub-family G member 4 (ABCG4) and prognosis in patients with non-small-cell lung cancer (NSCLC) remains unclear. NSCLC tissue samples (n = 140) and normal lung tissue samples (n = 90) were resected from patients with stage II to IV NSCLC between May 2004 and May 2009. ABCG4 mRNA and protein expressions were detected by RT-PCR, western blot, and immunohistochemistry. Patients received four cycles of cisplatin-based post-surgery chemotherapy and were followed up until May 31st, 2014. ABCG4 positivity rate was higher in NSCLC than in normal lung tissues (48.6% vs. 0%, P<0.001) and ABCG4 expression was significantly associated with poor differentiation, higher tumor node metastasis (TNM) stage, and adenocarcinoma histological type (all P<0.001). Univariate (HR = 2.284, 95%CI: 1.570-3.324, P<0.001) and multivariate (HR = 2.236, 95%CI: 1.505-3.321, P<0.001) analyses showed that ABCG4 expression was an independent factor associated with a poor prognosis in NSCLC. Patients with ABCG4-positive NSCLC had shorter median survival than ABCG4-negative NSCLC (20.1 vs. 43.2 months, P<0.001). The prognostic significance of ABCG4 expression was apparent in stages III and IV NSCLC. In conclusion, high ABCG4 expression was associated with a poor prognosis in patients with NSCLC treated with cisplatin-based chemotherapy.

EGFR mutations in surgically resected fresh specimens from 697 consecutive Chinese patients with non-small cell lung cancer and their relationships with clinical features.

We aimed to reveal the true status of epidermal growth factor receptor (EGFR) mutations in Chinese patients with non-small cell lung cancer (NSCLC) after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS). Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7%) patients had tyrosine kinase inhibitor (TKIs) sensitive EGFR mutations in 41 (14.5%) of the 282 squamous carcinomas, 155 (52.9%) of the 293 adenocarcinomas, 34 (39.5%) of the 86 adenosquamous carcinomas, one (9.1%) of the 11 large-cell carcinomas, 2 (11.1%) of the 18 sarcomatoid carcinomas, and 2 (28.6%) of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001), non-smokers (p = 0.047) and adenocarcinomas (p < 0.001). The rates of exon 19 deletion mutation (19-del), exon 21 L858R point mutation (L858R), exon 21 L861Q point mutation (L861Q), exon 18 G719X point mutations (G719X, including G719C, G719S, G719A) were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M) was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins) was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients.