[Floating-Harbor syndrome: a case report and literature review].

Floating-Harbor syndrome (FHS) is an autosomal dominant genetic disease caused by SRCAP mutation. This article reports the clinical features of a boy with FHS. The boy, aged 11 years and 7 months, attended the hospital due to short stature for more than 8 years and had the clinical manifestations of unusual facial features (triangularly shaped face, thin lips and long eyelashes), skeletal dysplasia (curvature finger), expressive language disorder, and retardation of bone age. Genetic detection revealed a novel heterozygous mutation, c.7330 C>T(p.R2444X), in the SRCAP gene. The boy was diagnosed with FHS based on these clinical manifestations and gene detection results. FHS is rare in clinical practice, which may lead to missed diagnosis and misdiagnosis, and gene detection may help with the clinical diagnosis of FHS in children.

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma.

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break-apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break-apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break-apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.

Malignant pleural effusion supernatants are substitutes for metastatic pleural tumor tissues in EGFR mutation test in patients with advanced lung adenocarcinoma.

BACKGROUND: Though the possibility of using malignant pleural effusions (MPEs) as alternatives for metastatic pleural tumor tissues (MPTTs) in epidermal growth factor receptor (EGFR) mutation test has been examined, due to the lack of studies comparing the results in matching MPEs and MPTTs, the clinical value of MPEs for advanced adenocarcinoma patients with pleural effusions is not confirmed. METHODS: EGFR mutation statuses in matching MPTTs, MPE supernatants and cell blocks, of 41 patients with advanced lung adenocarcinoma as diagnosed by thoracoscopy were analyzed using amplification refractory mutation system (ARMS). RESULTS: EGFR mutations were detected in 46.3% (19/41) of MPTTs, 43.9% (18/41) of MPE supernatants and 56.3% (18/32) of MPE cell blocks by ARMS analysis. Generally, the same EGFR statuses were identified in both MPTTs and matching MPE cell blocks of 81.3% patients (26/32), whereas MPTTs and matching MPE supernatants of 87.8% (36/41) patients shared the same EGFR status. Compared with EGFR mutation detection in MPTTs, the sensitivity of EGFR mutation detection in MPE-cell blocks was 87.5% (14/16), specificity was 75.0% (12/16), while the sensitivity of EGFR mutation detection in MPE-supernatants was 84.2% (16/19), specificity was 90.9% (20/22). CONCLUSIONS: The high concordance of EGFR mutation statuses between MPEs and MPTTs in lung adenocarcinoma patients with pleural metastasis as determined by ARMS analysis suggests that MPEs, particularly MPE supernatants, may be substitutes for MPTTs in EGFR mutation test.

Frequency, characterization, and prognostic analysis of PIK3CA gene mutations in Chinese esophageal squamous cell carcinoma.

PIK3CA gene mutations are found in numerous cancers but correlate differently with prognosis. Although the frequency of PIK3CA gene mutation in esophageal squamous cell carcinoma (ESCC) has been previously studied, a prognostic analysis has not been reported. Ninety-six surgically resected ESCC tissues were collected from Chinese patients and DNA was extracted. Gene mutations in PIK3CA (exons 9 and 20), EGFR (exons 18, 19, 20 and 21), KRAS (exons 2 and 3), and BRAF (exons 11 and 15) were screened using mutant-enriched liquid chip technology. PIK3CA gene mutations were identified in 12 of 96 ESCC cases (12.5%). No mutations were identified in EGFR, KRAS or BRAF genes in this study. Correlations between clinicopathological features and PIK3CA mutation status were analyzed and finally, patient survival information was used to determine the prognostic significance of PIK3CA mutation. Interestingly, the frequency of PIK3CA mutation was higher in female ESCC patients (31.3%, 5/16) than in males (8.8%, 7/80), and higher in patients with non-lymph node metastasis (19.6%, 10/51, P = .013) than in patients with lymph node metastasis (4.4%, 2/45, P = .025). Furthermore, patients with PIK3CA-mutated tumors showed a trend towards favorable overall survival (P = .085) but not disease-free survival (P = .238), suggesting that PIK3CA gene status may be a favorable predictive marker in ESCC patients.

Fibroblast growth factor receptor 4 Gly388Arg polymorphism in Chinese gastric cancer patients.

AIM: To investigate the contribution of the fibroblast growth factor receptor 4 (FGFR4) Gly388Arg polymorphism as a genetic risk factor for gastric cancer (GC) and to investigate any associations between this polymorphism and clinicopathological parameters and survival. METHODS: Tumors and matched adjacent non-cancer tissues were collected from 304 GC patients, and 5 mL of venous blood was collected from 62 GC patients and 392 age- and sex-matched healthy controls without cancer history from the same ethnic population. DNA was extracted, and direct sequencing analyses were performed to genotype the FGFR4 Gly388Arg polymorphism in all the samples. Differences in the genotype frequencies of the FGFR4 Gly388Arg polymorphism between GC patients and healthy controls were estimated using the χ(2) test. Binary logistic regression was used for all analysis variables to estimate risk as the ORs with 95%CIs. The relationships between the FGFR4 genotype and clinicopathological parameters were tested with the χ(2) test. The Kaplan-Meier product-limit method, the log-rank test, and the Cox regression model were applied to evaluate the effect of the FGFR4 genotype on the overall survival of patients with GC. RESULTS: In the present GC cohort, 118 patients (38.8%) were homozygous for the Gly388 allele, 124 patients (40.8%) were heterozygous, and 62 patients (20.4%) were homozygous for the Arg388 allele. The frequencies of the Gly/Gly, Gly/Arg, and Arg/Arg genotypes in the healthy controls were 33.6%, 48.0%, and 18.4%, respectively. The distributions of genotypes (χ(2) = 3.589, P = 0.166) and alleles (χ(2) = 0.342, P = 0.559) of the FGFR4 Gly388Arg polymorphism were not different between the GC patients and the healthy controls. Although we observed no correlation between the FGFR4 Gly388Arg polymorphism and clinicopathological parameters or survival in the total cohort of GC patients, the presence of the Arg388 allele was associated with shorter survival time in patients with GC if the tumor was small (log rank χ(2) = 5.449, P = 0.020), well differentiated (log rank χ(2) = 12.798, P = 0.000), T1 or T2 stage (log rank χ(2) = 4.745, P = 0.029), without lymph node involvement (log rank χ(2) = 6.647, P= 0.010), and at an early clinical stage (log rank χ(2) = 4.615, P = 0.032). CONCLUSION: Our results suggest that the FGFR4 Gly388Arg polymorphism is not a risk factor for GC cancer initiation but that it is a useful prognostic marker for GC patients when the tumor is relatively small, well differentiated, or at an early clinical stage.

The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.

AIMS: To evaluate the suitability of malignant pleural effusion (MPE) and plasma as surrogate samples for epidermal growth factor receptor (EGFR) mutation detection, and compare three different detection methods. METHODS: Matched tissue and plasma samples were collected from patients with advanced non-small cell lung cancer (NSCLC) (stage IIIB/IV adenocarcinoma/adenosquamous carcinoma), with matched MPE samples collected from a subgroup. DNA was extracted from tissue, MPE cell block, MPE supernatant and plasma before mutation detection by amplification refractory mutation system (ARMS) (all samples), Sanger sequencing and mutant-specific immunohistochemistry (IHC) (tissue and MPE cell blocks only). RESULTS: Sensitivity of MPE cell block, MPE supernatant and plasma versus tissue: 81.8% (9/11), 63.6% (7/11) and 67.5% (27/40); specificity was 80.0% (8/10), 100% (10/10) and 100% (46/46), respectively. Sensitivity of Sanger sequencing versus ARMS: 81.8% (27/33) for tissue, 40% (4/10) for MPE cell blocks; specificity was 100% (36/36 and 12/12) for both. Sensitivity of mutant-specific IHC versus ARMS: 54.8% (17/31) for tissue, 50.0% (6/12) for MPE cell blocks; specificity was 97.1% (34/35) and 100% (14/14), respectively. CONCLUSIONS: MPE and plasma are valid surrogates for NSCLC tumour EGFR mutation detection when tissue is not available. ARMS is most suitable for mutation detection in tissue and MPE cell blocks; however, mutant-specific IHC could be a complementary method when DNA-based molecular testing is unavailable.

Comparison of different methods for detecting epidermal growth factor receptor mutations in peripheral blood and tumor tissue of non-small cell lung cancer as a predictor of response to gefitinib.

BACKGROUND: Previous studies have reported that epidermal growth factor receptor (EGFR) mutation in tumor tissue and peripheral blood can predict the response to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). However, the heterogeneity of the sample sources makes it difficult to evaluate the detecting methodologies. The goal of this study is to compare different methods for analyzing EGFR mutation in blood and tumor tissue. MATERIALS AND METHODS: Fifty-one advanced NSCLC patients treated with gefitinib were included in the study. The EGFR mutation status of each patients' blood was analyzed by denaturing high-performance liquid chromatography (DHPLC), mutant-enriched liquidchip (ME-Liquidchip), and Scorpion Amplification Refractory Mutation System (Scorpion-ARMS) kits. EGFR mutation information in paired tumor samples detected by Scorpion-ARMS served as a reference. Comparative analyses were performed on mutation status results obtained from different methods and on the association between the clinical outcome of TKI treatment and EGFR mutation status. RESULTS: The response rate (RR) in the whole group was 33.3%. EGFR mutation rates were identified as 15.7%, 27.5%, and 29.4% by DHPLC, ME-Liquidchip, and Scorpion-ARMS in blood, respectively. In 34 cases that had paired tumor samples, the mutation rate in tissue was 41.2%. The RRs of patients with mutation detected by different methods were 71.4% (tumor), 62.5% (blood, DHPLC), 50.0% (blood, ME-Liquidchip), and 66.7% (blood, Scorpion-ARMS). EGFR mutation detected by Scorpion-ARMS in blood and tumor tissues had better prediction of RR to EGFR-TKI (P = 0.002 and P = 0.001) than mutation detected with DHPLC and ME-Liquidchip. CONCLUSION: Tumor tissue sample is the best source for EGFR mutation analysis in NSCLC patients. Peripheral blood samples may be used as an alternative source only in special conditions. Scorpion-ARMS, DHPLC, or ME-Liquidchip methods are all optional for detecting tumor EGFR mutation from blood.

A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis.

PURPOSE: Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. MATERIALS AND METHODS: The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. RESULTS: MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (≥ 202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. CONCLUSION: An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.

KRAS and BRAF mutations in prostate carcinomas of Chinese patients.

Aberrance in the RAS/RAF/MEK/MAPK pathway is reportedly important in many tumor types, including prostate carcinoma. Continuous activation of the pathway can be caused by mutations of upstream targets such as KRAS and BRAF. However, rates of KRAS and BRAF mutations in prostate carcinoma are reported to vary in different populations. To date, there has been no such report in Chinese patients. In this study, we examined 121 samples of prostate carcinoma in Chinese subjects for mutations at codons 12 and 13 of KRAS and codon 600 of BRAF by means of the mutant-enriched polymerase chain reaction-coupled sequencing method. The identified KRAS and BRAF mutations were analyzed for association with tumor differentiation and clinical stage. The result showed that KRAS mutations were detected in 9.1% (11 of 121) of prostate carcinomas, while no BRAF mutation was found in any case studied. No association was found between KRAS mutation and clinicopathological characteristics of the tumors. Our study suggests that mutations of KRAS, not BRAF, may play a role in the pathogenesis of prostate carcinoma in Chinese patients.

Methylation changes of H19 gene in sperms of X-irradiated mouse and maintenance in offspring.

The nature of imprinting is just differential methylation of imprinted genes. Unlike the non-imprinted genes, the methylation pattern of imprinted genes established during the period of gametogenesis remains unchangeable after fertilization and during embryo development. It implies that gametogenesis is the key stage for methylation pattern of imprinted genes. The imprinting interfered by exogenous factors during this stage could be inherited to offspring and cause genetic effect. Now many studies have proved that ionizing irradiation could disturb DNA methylation. Here we choose BALB/c mice as a research model and X-ray as interfering source to further clarify it. We discovered that the whole-body irradiation of X-ray to male BALB/c mice could influence the methylation pattern of H19 gene in sperms, which resulted in some cytosines of partial CpG islands in the imprinting control region could not transform to methylated cytosines. Furthermore, by copulating the interfered male mice with normal female, we analyzed the promoter methylation pattern of H19 in offspring fetal liver and compared the same to the pattern of male parent in sperms. We found that the majority of methylation changes in offspring liver were related to the ones in their parent sperms. Our data proved that the changes of the H19 gene methylation pattern interfered by X-ray irradiation could be transmitted and maintained in the first-generation offspring.

Prenatal fetal sex diagnosis by detecting amelogenin gene in maternal plasma.

OBJECTIVES: To provide a new, reliable noninvasive method for fetal sex determination. METHODS: Fetal sex was detected in 32 early pregnant women by identifying the amelogenin gene in maternal plasma using nested PCR analysis. First, the 122/128 bp of X-Y homologous region containing 6 bp deletions in the intron 3 of amelogenin gene in X chromosome was amplified, and then the nested PCR was carried out, whose 3' end of the upstream primer is just located in the deletion region. The fetus was male or female, depending on whether it had the 89-bp nested PCR product or not. RESULTS: The 89 bp of nested PCR product was detected in 19 plasma samples obtained from pregnant women, deducing they bear the male fetus and the remaining pregnant women bear female. When compared with the birth outcome, two samples were pseudo-positive. The coincidence was 93.8%. This method had high sensitivity that even trace amount of target fetal DNA (10 pg) could be detected. CONCLUSIONS: This conventional nested PCR analysis of amelogenin gene promises to be a reliable method for noninvasive fetal sex determination at early pregnancy using maternal plasma DNA.