Search

Objective We present detection of hypermethylation at H19 differentially methylated region (DMR) at amniocentesis in a fetus with overgrowth, distended abdomen and Beckwith-Wiedemann syndrome (BWS). Case report A 31-year-old, gravida 2, para 1, woman was referred for genetic counseling at 22 weeks of gestation because of fetal overgrowth with fetal biometry equivalent to 24 weeks of gestation and a distended abdomen with an abdominal circumference equivalent to 26 weeks of gestation. She did not undergo any assisted reproductive technology during this pregnancy. Amniocentesis was performed at 23 weeks of gestation. Conventional cytogenetic analysis revealed a karyotype of 46,XX. Array comparative genomic hybridization analysis on the DNA extracted from uncultured amniocytes revealed no genomic imbalance. Methylation analysis on the DNA extracted from amniocytes revealed hypermethylation at H19DMR [imprinting center 1 (IC1)] and normal methylation at KvDMR1 (IC2). The methylation test confirmed the diagnosis of BWS in the fetus. The parents decided to continue the pregnancy. At 36 weeks of gestation, a 4000-g female baby was delivered with macroglossia, ear tags and creases, and an enlarged liver, consistent with the phenotype of BWS. Conclusion Prenatal diagnosis of fetal overgrowth should include a differential diagnosis of BWS, and methylation analysis of H19DMR (IC1) and KvDMR1 (IC2) is useful under such a circumstance.

Objective The Lauren classification is an important histologic classification of gastric cancer (GC) with different biological behaviors between histologic types. Background To date, there are few reports on the genetic alterations and survival differences between different histologic types according to the Lauren classification. Methods In total, 433 GC patients undergoing surgery were enrolled. The clinicopathologic features, prognoses, and genetic alterations of the different Lauren types were compared. Results Diffuse-type GC was associated with a younger age, female predominance, more Borrmann type 3 and 4 tumors, more advanced pathologic tumor (T) and node (N) categories, more tumor recurrences (especially peritoneal recurrence), and worse 5-year overall survival and disease-free survival rates than intestinal-type GC and mixed-type GC. Regarding genetic alterations, mixed-type GC was associated with more TP53 mutations than intestinal-type GC and diffuse-type GC. Multivariate analysis demonstrated the following independent prognostic factors: age, Lauren classification, and pathologic T and N categories. Regarding mixed-type GC, diffuse-type major tumors were associated with more lymphovascular invasion, a more advanced N category and tumor, node, metastasis stage, and fewer PI3K/AKT pathway mutations than intestinal-type major tumors. Conclusions Diffuse-type GC had unfavorable clinicopathologic features and a worse prognosis than intestinal-type GC. For mixed-type GC, the clinicopathologic features and genetic alterations were different between intestinal-type major tumors and diffuse-type major tumors.

To evaluate the feasibility of incorporating genetic screening for neonatal intrahepatic cholestasis, caused by citrin deficiency (NICCD), into the current newborn screening (NBS) program. We designed a high-throughput iPLEX genotyping assay to detect 28 SLC25A13 mutations in the Chinese population. From March 2018 to June 2018, 237 630 newborns were screened by tandem mass spectrometry at six hospitals. Newborns with citrulline levels between 1/2 cutoff and cutoff values of the upper limit were recruited for genetic screening using the newly developed assay. The sensitivity and specificity of the iPLEX genotyping assay both reached 100% in clinical practice. Overall, 29 364 (12.4%) newborns received further genetic screening. Five patients with conclusive genotypes were successfully identified. The most common SLC25A13 mutation was c.851_854del, with an allele frequency of 60%. In total, 658 individuals with one mutant allele were identified as carriers. Eighteen different mutations were observed, yielding a carrier rate of 1/45. Notably, Quanzhou in southern China had a carrier rate of up to 1/28, whereas Jining in northern China had a carrier rate higher than that of other southern and border cities. The high throughput iPLEX genotyping assay is an effective and reliable approach for NICCD genotyping. The combined genetic screening could identify an additional subgroup of patients with NICCD, undetectable by conventional NBS. Therefore, this study demonstrates the viability of incorporating genetic screening for NICCD into the current NBS program.

Background The form of microsatellite instability (MSI) affecting tetranucleotide repeats known as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has emerged as a new potential biomarker in multiple cancers. In colorectal cancer (CRC), the correlation between EMAST and MSI mutations remain inconclusive. Materials and Methods We evaluated 1,505 patients with CRC using five EMAST markers (D20S82, D20S85, D8S321, D9S242, and MYCL1) and the Bethesda panel of MSI markers. Most commonly, mutations involved in CRCs were identified by MassArray Assay, and DNA repair genes were analyzed by next-generation sequencing. Clinical characteristics and prognostic relevance were correlated with EMAST and MSI. Results Tumors that were EMAST positive and MSI high (MSI-H) were detected in 159 (10.6%) and 154 (10.2%) of 1,505 patients with CRC. Patients were divided into four groups according to EMAST and MSI status (EMAST-positive and MSI-H, EMAST-positive and microsatellite-stable [MSS], EMAST-negative and MSI-H, and EMAST-negative and MSS). The EMAST-positive and MSI-H group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Furthermore, compared with only EMAST-positive tumors or only MSI-H tumors, tumors that were both EMAST-positive and MSI-H had a higher frequency of MLH1, MSH3, MSH6, PMS2, and EXO1 gene mutations. Finally, the presence of EMAST-positive and MSI-H tumors was a good prognostic indicator in CRC. Conclusion High mutations in several DNA repair genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy. Implications for Practice Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a unique molecular subtype of colorectal cancer (CRC). The current study demonstrated that the EMAST-positive and MSI-high (MSI-H) group was associated with female predominance, higher prevalence of proximal colon tumors, early stage tumors, poorly differentiated tumors, mucinous histology, and higher incidence of mutations in PI3KCA, BRAF, TGFBR, PTEN, and AKT1 compared with other groups. Most importantly, high mutations in DNA repair genes and MSI-related genes in EMAST-positive and MSI-H tumors suggest that this subtype of CRC might be more suitable for treatment with immune therapy compared with MSI-H tumors alone.

Background Helicobacter pylori (HP) can induce epithelial cells and intestinal metaplasia with genetic damage that makes them highly susceptible to the development of gastric cancer (GC). Materials and Methods Between 2005 and 2010, 356 patients with gastric cancer who received curative surgery were enrolled. Analysis of HP, Epstein-Barr virus (EBV) infection, PIK3CA amplification, and mutation analysis of 68 mutations in eight genes using a mass spectrometric single-nucleotide polymorphism genotyping technology was conducted. The clinicopathological characteristics of patients with or without HP infection were compared. Results Among the 356 patients, 185 (52.0%) had HP infection. For intestinal-type GC, patients with HP infection were more likely to be younger and had fewer PI3K/AKT pathway genetic mutations than those without HP infection. For diffuse-type GC, patients with HP infection were characterized by less male predominance, less lymphoid stroma, fewer microsatellite instability-high tumors, and fewer PI3K/AKT pathway genetic mutations than those without HP infection. Patients with HP infection had less tumor recurrence and a better 5-year overall survival (87.7% vs. 73.9%, p = .012) and disease-free survival (64.1% vs. 51.3%, p = .013) than those without HP infection, especially for intestinal-type GC. For EBV-negative GC, patients with HP infection had fewer PI3K/AKT pathway mutations and a better 5-year overall survival and disease-free survival than those without HP infection. Multivariate analysis demonstrated that HP infection was an independent prognostic factor regarding overall survival and disease-free survival. Conclusion Patients with GC with HP infection were associated with fewer PI3K/AKT pathway genetic mutations and better survival than those without HP infection, especially for EBV-negative and intestinal-type GC. Implications for Practice Patients with gastric cancer with Helicobacter pylori (HP) infection had fewer PI3K/AKT pathway genetic mutations, less tumor recurrence, and better survival than those without HP infection, especially for Epstein-Barr virus (EBV)-negative and intestinal-type gastric cancer. HP infection is an independent prognostic factor regarding overall survival and disease-free survival. Future in vivo and in vitro studies of the correlation among HP infection, PI3K/AKT pathway, and EBV infection in gastric cancer are required.

BackgroundThe prognosis of mucinous adenocarcinoma (MAC) and non-mucinous adenocarcinoma (NMAC) in colorectal cancer (CRC) is controversial, and the molecular differences between them are unclear.MethodsBetween 2000 and 2010, a total of 1,483 CRC patients were included. Among them, 73 patients (4.9%) were diagnosed with MAC. The clinicopathological features and genetic alterations were compared between MAC and NMAC.ResultsAfter propensity score matching to balance age and sex between MAC and NMAC patients, 292 CRC patients (73 MAC and 219 NMAC) were enrolled in the analysis at a 1:3 ratio. In right-sided colon cancer, patients with MAC were more likely to have Borrmann types 3 and 4 tumors, poor differentiation, and advanced T category and tumor, node, metastasis (TNM) stage, chemotherapy, and a similar 5-year overall survival (OS) rate compared with patients with NMAC. In left-sided colon cancer and rectal cancer, patients with MAC were more likely to have Borrmann types 3 and 4 tumors, poor differentiation, lymphovascular invasion, advanced T and N categories and TNM stages, chemotherapy, and a worse 5-year OS rate than patients with NMAC. Regarding genetic alterations, for NMAC, right-sided colon cancer had more BRAF mutations than left-sided colon cancer and rectal cancer. For MAC, right-sided colon cancer was associated with more microsatellite instability-high tumors and more AKT1 mutations than left-sided colon cancer and rectal cancer.ConclusionThe genetic alterations are distinct between MAC and NMAC in CRC. Tumor location may have an impact on genetic alterations and patient prognosis in MAC and NMAC.

The molecular difference between synchronous and metachronous metastases in colorectal cancer (CRC) remains unclear. Between 2000 and 2010, a total of 492 CRC patients were enrolled, including 280 with synchronous metastasis and 212 with metachronous metastasis. Clinicopathological and molecular features were compared between the two groups. Patients with synchronous metastasis were more likely to have right-sided CRC, poorly differentiated tumors, lymphovascular invasion, advanced pathological tumor (T) and node (N) categories, and liver metastases than those with metachronous metastasis. For right-sided CRC, patients with synchronous metastasis had more lymphovascular invasion and liver metastases than those with metachronous metastasis. For left-sided CRC, patients with synchronous metastasis were more likely to have poorly differentiated tumors, lymphovascular invasion, advanced pathological T and N categories, and liver metastases than those with metachronous metastasis. Regarding the genetic mutations, patients with metachronous metastasis had more mutations in TP53, NRAS, and HRAS and fewer mutations in APC than those with synchronous metastasis; for right-sided CRC, synchronous metastasis was associated with more APC mutations than metachronous metastasis, while for left-sided CRC, metachronous metastasis was associated with more TP53 and NRAS mutations than synchronous metastasis. The 5-year overall survival (OS) rates were significantly higher in metachronous metastasis patients than in synchronous metastasis patients, especially those with left-sided CRC. Multivariate analysis showed that age, sex, lymphovascular invasion, pathological N category, metachronous metastasis, and BRAF and NRAS mutations were independent prognostic factors affecting OS. CRC patients with synchronous metastasis had a worse OS than those with metachronous metastasis and exhibited distinct genetic mutations.

Few reports have investigated different genetic alterations according to age in various cancers. In total, 1749 GC patients receiving curative surgery were enrolled. The clinicopathological features, and prognoses were compared between younger (

Objective Immune checkpoint inhibitors (ICIs) have become the standard of care in various cancers, although the predictive tool is still unknown. Methods This study aimed to develop a novel gene panel by selecting DNA damage response (DDR) genes from the Catalogue of Somatic Mutations in Cancer (COSMIC) databank and validating them in previously reported cohorts. This association between DDR gene mutations and tumor mutation burden or microsatellite status was analysed from The Cancer Genome Atlas (TCGA) databank. Furthermore, we made the gene panel clinically accessible and predicted the response in clinical patients receiving ICIs by using cell‐free DNA. Results The top 20 mutated DDR genes in various cancers (total 37 genes) were taken from the COSMIC databank, and the DDR genes found to individually predict a response rate > 50% in Van Allen's cohort were selected (Science, 350, 2015 and 207). Eighteen DDR genes were selected as the gene panel. The prevalence and predicted response rate were validated in the other three reported cohorts. Tumor mutational burden‐high was positively associated with mutations of the 18 DDR genes for most cancers. We used cell‐free DNA to test the DDR gene panel and validated by our patients receiving ICIs. This DDR gene panel accounted for approximately 30% of various cancers, achieving a predicted response rate of approximately 60% in patients with a mutated gene panel receiving ICIs. Conclusion This gene panel is a novel and reliable tool for predicting the response to ICIs in cancer patients and guides the appropriate administration of ICIs in clinical practice., Immune checkpoint inhibitors (ICIs) have been widely used to treat various cancers and have achieved impressive success, resulting in a new era of anticancer therapy. However, only a subset of patients experienced lasting responses; therefore, exploring predictive biomarkers is critical to identify the patients who have benefit from ICIs. This gene panel using liquid biopsy is one of the best predictive biomarkers presently available and can help physicians to predict the response to ICIs and judge the utility of ICIs in clinical practice.

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disorder and the leading genetic cause of infant mortality. Early detection of SMA through newborn screening (NBS) is essential to selecting pre-symptomatic treatment and ensuring optimal outcome, as well as, prompting the urgent need for effective screening methods. This study aimed to determine the feasibility of applying an Agena iPLEX SMA assay in NBS for SMA in China. Methods: We developed an Agena iPLEX SMA assay based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and evaluated the performance of this assay through assessment of 167 previously-genotyped samples. Then we conducted a pilot study to apply this assay for SMA NBS. The SMN1 and SMN2 copy number of screen-positive patients were determined by multiplex ligation-dependent probe amplification analysis. Results: The sensitivity and specificity of the Agena iPLEX SMA assay were both 100%. Three patients with homozygous SMN1 deletion were successfully identified and conformed by multiplex ligation-dependent probe amplification analysis. Two patients had two SMN2 copies, which was correlated with severe SMA type I phenotype; both of them exhibited neurogenic lesion and with decreased muscle power. Another patient with four SMN2 copies, whose genotype correlated with milder SMA type III or IV phenotype, had normal growth and development without clinical symptoms. Conclusions: The Agena iPLEX SMA assay is an effective and reliable approach for population-based SMA NBS. The first large-scale pilot study using this assay in the Mainland of China showed that large-scale implementation of population-based NBS for SMA is feasible.

Background Aberrant DNA methylation is involved in gastric carcinogenesis and may serve as a useful biomarker in the diagnosis and detection of gastric cancer (GC) recurrence. Results A total of 157 patients who received surgery for GC were enrolled in the present study. A genome-wide methylation analysis was performed in tumor and adjacent normal tissues for the discovery set of 16 GC patients; the top three hypermethylated CpG sites of DNA promoters were selected for validation in tissue and plasma samples for the validation set of 141 GC patients. The frequencies of the top three hypermethylated genes in available patient tissues (n = 141) and plasma samples (n = 106) were 41.8% and 38.7%, respectively, for ADAM19; 40.4% and 42.5%, respectively, for FLI1; and 56.7% and 50.9%, respectively, for MSC. In both tissue and plasma samples, FLI1 hypermethylation was associated with more advanced GC and liver and distant lymphatic metastasis, and ADAM19 hypermethylation was associated with more stage IV GC. In plasma samples, MSC hypermethylation was more common in non-superficial type GC than samples without MSC hypermethylation. In both tissue and plasma samples, patients with methylation of all the three genes had significantly more liver metastases, distant lymphatic metastases, and paraaortic lymph node metastases than patients with two or fewer hypermethylated genes. The survival analysis showed that only for stage III GC, patients with hypermethylation of two or three genes had a worse 5-year disease-free survival rate than those with hypermethylation of one or none of the three genes. Subgroup analysis showed that FLI1 hypermethylation in both tissue and plasma samples was associated with liver metastasis in MSI−/EBV− GC, and MSC hypermethylation in tissue samples was correlated with liver metastasis in MSI+ or EBV+ GC. Patients with FLI1 hypermethylation in plasma samples had a significantly worse 5-year disease-free survival rate than those without FLI1 hypermethylation in MSI−/EBV− GC. FLI1 hypermethylation was an independent prognostic factor affecting the overall survival and disease-free survival in both tissue and plasma samples. Conclusions DNA methylation is a useful biomarker for predicting tumor recurrence patterns and GC patient survival.

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5. Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC (P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression (P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set (P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients (P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC.

Background We assumed that targeted next‐generation sequencing (NGS) of mismatch repair‐associated genes could improve the detection of driving mutations in colorectal cancers (CRC) with microsatellite instability (MSI) and microsatellite alterations at selected tetranucleotide repeats (EMAST) and clarify the somatic mutation patterns of CRC subtypes. Material and methods DNAs from tumors and white blood cells were obtained from 81 patients with EMAST(+)/MSI‐high (MSI‐H), 78 patients with EMAST(+)/microsatellite stable (MSS), and 72 patients with EMAST(−)/MSI‐H. The germline and somatic mutations were analyzed with a 16‐genes NGS panel. Results In total, 284 germline mutations were identified in 161 patients. The most common mutations were in EPCAM (24.8%), MSH6 (24.2%), MLH1 (21.7%), and AXIN2 (21.7%). Germline mutations of AXIN2, POLE, POLD1, and TGFBR2 also resulted in EMAST and MSI. EMAST(+)/MSI‐H tumors had a significant higher mutation number (205.9 ± 95.2 mut/MB) than tumors that were only EMAST(+) or MSI‐H (118.6 ± 64.2 and 106.2 ± 54.5 mut/MB, respectively; both P, For patients with microsatellite instability or microsatellite alterations at selected tetranucleotide repeats, the somatic mutation burden occurs due to the dysfunction of downstream effectors but not the affected gene with germline mutation. The dysfunction of AXIN2 might affect both the canonical APC pathway and the hypermutation mechanism.

Additional file 6: Table S2. The frequency of methylation of the three genes in tissue and plasma samples according to the status of MSI/EBV.

BACKGROUND. Helicobacter pylori (HP) can induce epithelial cells and intestinal metaplasia with genetic damage that makes them highly susceptible to the development of gastric cancer (GC). MATERIALS AND METHODS. Between 2005 and 2010, 356 patients with gastric cancer who received curative surgery were enrolled. Analysis of HP, Epstein‐Barr virus (EBV) infection, PIK3CA amplification, and mutation analysis of 68 mutations in eight genes using a mass spectrometric single‐nucleotide polymorphism genotyping technology was conducted. The clinicopathological characteristics of patients with or without HP infection were compared. RESULTS. Among the 356 patients, 185 (52.0%) had HP infection. For intestinal‐type GC, patients with HP infection were more likely to be younger and had fewer PI3K/AKT pathway genetic mutations than those without HP infection. For diffuse‐type GC, patients with HP infection were characterized by less male predominance, less lymphoid stroma, fewer microsatellite instability‐high tumors, and fewer PI3K/AKT pathway genetic mutations than those without HP infection. Patients with HP infection had less tumor recurrence and a better 5‐year overall survival (87.7% vs. 73.9%, p = .012) and disease‐free survival (64.1% vs. 51.3%, p = .013) than those without HP infection, especially for intestinal‐type GC. For EBV‐negative GC, patients with HP infection had fewer PI3K/AKT pathway mutations and a better 5‐year overall survival and disease‐free survival than those without HP infection. Multivariate analysis demonstrated that HP infection was an independent prognostic factor regarding overall survival and disease‐free survival. CONCLUSION. Patients with GC with HP infection were associated with fewer PI3K/AKT pathway genetic mutations and better survival than those without HP infection, especially for EBV‐negative and intestinal‐type GC. IMPLICATIONS FOR PRACTICE. Patients with gastric cancer with Helicobacter pylori (HP) infection had fewer PI3K/AKT pathway genetic mutations, less tumor recurrence, and better survival than those without HP infection, especially for Epstein‐Barr virus (EBV)‐negative and intestinal‐type gastric cancer. HP infection is an independent prognostic factor regarding overall survival and disease‐free survival. Future in vivo and in vitro studies of the correlation among HP infection, PI3K/AKT pathway, and EBV infection in gastric cancer are required.

Rationale Paragangliomas (PGLs) are rare neuroendocrine tumors that are strongly influenced by genetics, and succinate dehydrogenase-deficient PGLs appear to constitute one of the most important categories. Interestingly, somatic PGLs only possess genomic alterations involving the SDHB and SDHD subunits, and no SDHA alterations have been described. Here, we are presenting the clinical and genetic analyses of 2 cases with the first somatic SDHA variant identified in PGLs. Patient concerns Here, we reported 2 family members with the diagnosis of PGL. Patient 1 is a 55-year-old woman with a functionally perigastric PGL that co-occurred with a gastric gastrointestinal stromal tumor (GIST), and patient 2 is a 43-year-old woman with a nonfunctionally pericardial PGL, who was the younger sister of the first patient. Diagnoses Imaging surveys of the 2 cases depicted the presence of a perigastric and a pericardial mass, respectively. A diagnosis of paragangliomas was established by immunohistochemistry (IHC). Interventions Both patients underwent single-stage resection of the lesion after preoperative oral α-adrenoceptor therapy for 2 weeks. We later performed comprehensive genomic profiling on the tumor samples, including PGL and GIST from patient 1 and PGL from patient 2, and searched for novel actionable mutations, including in all succinate dehydrogenase subunits, as the IHC results were negative for SDHB. Outcomes Both patients had an uneventful recovery after surgery and the sequencing showed a novel somatic variant in the SDHA gene on chromosome 5q11 (c.1945_1946delTT). Regular follow-up with biochemical testing and image studies showed no evidence of recurrence after a year for patient 1 and 6 years for patient 2. Lessons PGLs often lead to considerable diagnostic difficulty due to their multiple anatomical locations and variable symptoms, as presented by our cases. The comprehensive use of images and plasma/urine catecholamine measurement can aid the diagnosis of PGLs. In addition, our findings also demonstrate the usefulness and importance of genetic analysis of SDHA mutations in patients exhibiting SDHB IHC-negative PGL. Additional studies utilizing comprehensive genomic profiling are needed to identify the group of PGLs harboring this SDHA genomic alteration.

With the progression of molecular techniques, the detection of circulating plasma DNA (cpDNA) is clinically feasible. However, the role of the cpDNA levels in gastric cancer is not well understood. This study assessed the mutational profile in primary tumors and clarified the clinical utility of quantitative and qualitative cpDNA alterations in 277 patients with advanced gastric cancer. The concentrations of cpDNA were measured by TaqMan qPCR, and 68 mutations in 8 genes were studied for cpDNA mutations. The median cpDNA concentrations in patients with stages I, II, and III gastric cancer were 3979, 3390 and 4278 copies/mL, respectively, and increased to 11,380 copies/mL in patients with Stage IV gastric cancer (p < 0.001). Among the 35 patients harboring cpDNA mutations, Stage IV patients (100%) were more likely to display high cpDNA levels than were Stage I (33.3%), II (75%) and III patients (66.7%) (p = 0.037). Patients displaying high cpDNA levels were more likely to experience peritoneal recurrence and exhibited significantly lower 5-year overall survival rates (39.2% vs. 45.8%, p = 0.039) than did patients displaying low cpDNA levels. Only for late stage (Stages III or IV) gastric cancer, patients harboring cpDNA mutations were more likely to experience vascular invasion (20% vs. 2.4%, p = 0.036) and exhibited a lower 5-year overall survival rate than did those lacking cpDNA mutations (5.6% vs. 31.5%, p = 0.028). High cpDNA levels are associated with peritoneal recurrence and poor prognosis in patients with advanced gastric cancer; harboring cpDNA mutations is associated with poor prognosis among patients with late stage gastric cancer.

// Wen-Liang Fang 1, 2 , Kuo-Hung Huang 1, 2, 3 , Yuan-Tzu Lan 2, 4 , Chien-Hsing Lin 5 , Shih-Ching Chang 2, 4, * , Ming-Huang Chen 2, 6 , Yee Chao 2, 6 , Wen-Chang Lin 7, 8 , Su-Shun Lo 2, 9 , Anna Fen-Yau Li 2, 10 , Chew-Wun Wu 1, 2 , Shih-Hwa Chiou 3, 11, 12 , Yi-Ming Shyr 1, 2, * 1 Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei City, Taiwan 2 School of Medicine, National Yang-Ming University, Taipei City, Taiwan 3 Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei City, Taiwan 4 Division of Colon & Rectal Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei City, Taiwan 5 Genome Research Center, National Yang-Ming University, Taipei City, Taiwan 6 Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 7 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 8 Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei, Taiwan 9 National Yang-Ming University Hospital, Yilan City, Taiwan 10 Department of Pathology, Taipei Veterans General Hospital, Taipei City, Taiwan 11 Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei City, Taiwan 12 Institute of Pharmacology, National Yang-Ming University, Taipei City, Taiwan * These authors have contributed equally to this work Correspondence to: Shih-Ching Chang, e-mail: changsc@vghtpe.gov.tw Yi-Ming Shyr, e-mail: ymshyr@vghtpe.gov.tw Keywords: PI3K/AKT pathway, PIK3CA amplifications, recurrence pattern, diffuse-type, prognosis Received: August 08, 2015 Accepted: December 05, 2015 Published: December 17, 2015 ABSTRACT Mutations in genes involved in the PI3K/AKT pathway and amplifications of the PIK3CA gene in gastric cancer and their associations with clinicopathological characteristics and EBV infection were analyzed in this study. A total of 431 patients with gastric adenocarcinomas were enrolled, and 39 mutation hotspots were evaluated in these patients using MALDI-TOF mass spectrometry were analyzed. PIK3CA amplifications were analyzed using real-time quantitative PCR. Regarding patients with intestinal-type gastric cancer, those with mutations in PI3K/AKT pathway genes were also more likely to have tumors located in the lower-third of the stomach than were those without mutations. Regarding patients with diffuse-type gastric cancer, those with PI3K/AKT pathway mutations were more likely to have tumors located in the upper-third of the stomach and to have more hematogenous metastases, particularly in the liver and lungs, than were patients without such mutations (22.2% vs. 4.5%). No significant survival difference was observed between patients with vs. without PI3K/AKT pathway mutations. Mutations in PI3K/AKT pathway genes were associated with hematogenous metastasis in patients with diffuse-type gastric cancer. Only when the tumors were located in the middle-third of stomach, tumor with mutations of the PIK3CA gene or mutations of the PI3K/AKT pathway genes were associated with more EBV infection than those without mutations. Patients with PIK3CA amplifications were more likely to have diffuse-type and poorly differentiated gastric cancers and were more likely to experience peritoneal recurrence compared with those without PIK3CA amplifications. Even upon subgroup analysis, PI3KCA amplifications were found to not affect the patients’ outcomes.

e15052 Background: Immune checkpoint inhibitors (ICIs) have become the standard of care in various cancers, although predictive biomarkers for efficacy still remain largely unknown. Methods: This study aims to develop a novel gene panel by selecting DNA damage and repair (DDR) genes from the Catalogue of Somatic Mutations in Cancer (COSMIC) database and perform validation using publicly available cohorts. Association between the DDR gene panel and tumor mutation burden or microsatellite status were analyzed from The Cancer Genome Atlas (TCGA) database. To further improve the clinical accessibility of the DDR gene panel, we analyzed the feasibility of its predictive value using cell free DNA in from 64 patients receiving ICIs. Results: The 193 mutated DDR genes in various cancers were chosen from the COSMIC databank, and the DDR genes found to individually predict a response rate > 50% in publicly melanoma cohort (Van Allen, et al) were selected. Eighteen DDR genes were selected as the DDR gene panel. The prevalence of the DDR gene mutations and predicted response rate of ICIs were validated in other three publicly available cohorts (Hugo et al, Synder et al, and Rizvi et al.). Tumor mutational burden and high microsatellite instability were positively associated with mutations of the 18 DDR genes for most cancers. Further validation was performed using cell-free DNA from cancer patients receiving ICIs. This DDR gene panel was detected in approximately 30% of various cancers, achieving a predicted response rate of approximately 60% in patients receiving ICIs. Conclusions: Our developed DDR gene panel can serve as a novel and reliable biomarker that can provide critical information for predicting the response to ICIs in cancer patients and guide the appropriate administration of ICIs in clinical practice.

Background Colorectal cancer stem cells (CSCs) serve crucial functions in tumor relapse, metastasis and therapy failure. Interleukin 17 receptor A (IL-17RA) is a potent mediator in the pathogenesis and progression of colorectal cancer. Our previous study showed that IL-17RA could promote angiogenesis, tumor growth and metastasis. In addition, IL-17RA was correlated with CRC recurrence. Recent study showed IL-17RA play a potential role in self-renewal in glioma stem cells. This study aims to evaluate the functional role and mechanism of IL-17RA in colorectal CSCs. Methods In this study, IL-17RA stable overexpression cells were used to examine the change of sphere formation, proliferation, CSC markers and epithelial-mesenchymal transition biomarkers and the regulation mechanism of signaling pathway. The specific IL-17RA signaling inhibitors were used to evaluate the function of the IL-17RA signaling affecting the CSC markers, epithelial-mesenchymal transition gene expression and sphere formation. Results We observed high IL-17RA expression can significantly promote the self-renewal by increasing stem cell markers CD133, ALDH1, Lgr5 and Sox2 expression and the ability to form tumorsphere in SW480 and SW620 cells. In additional, IL-17RA overexpression markedly increased chemoresistant ability and mesenchymal markers Vimentin, Slug, Snail, Zeb1expression. The STAT3 inhibitor remarkably decreased the CD133 stem cell gene, drug resistance and tumorsphere ability. Conclusions IL-17RA can enhance the self-renewal ability via activating the STAT3 pathway. Therefore, we expect that IL-17RA could serves as a prognosis marker and a potential therapeutic target in CRC. Legal entity responsible for the study Chih-Yung Yang. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background and objectives Identification of mutations in the downstream epidermal growth factor receptor (EGFR) signaling pathway could provide important insights of EGFR-targeted therapies in colorectal cancers. We analyzed the mutation spectra of the PI3K/PTEN/AKT and RAS/RAF/MAPK pathways in colorectal cancers and the associations of these mutations with sites of metastases or recurrence. Methods The study population comprised 1,492 retrospectively collected stages I–IV colorectal cancer specimens. Tissue was obtained between 2000 and 2010 at a single hospital. We analyzed 61 hot spots using MALDI–TOF mass spectrometry for nucleic acid analysis. Results Mutations were found in the RAS pathway in 47.3% of patients and in the PI3K pathway in 14.3% of patients, with 9.2% of patients carrying mutations in both pathways. Both the RAS and PI3K pathway mutations were significantly associated with proximal tumors, mucinous tumors, and microsatellite instability. Tumors carrying a RAS pathway mutation exhibited a higher frequency of lung and peritoneal metastasis than did tumors with a wild-type gene (P = 0.025 and 0.009, respectively). NRAS gene mutation was significantly associated with lung metastasis (P = 0.001). Conclusions Somatic mutations in the RAS pathway of the primary tumor in colorectal cancer can influence patterns of metastasis and recurrence. J. Surg. Oncol. 2015 111:905–910. © 2014 Wiley Periodicals, Inc.

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5. Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC (P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression (P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set (P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients (P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC.

// Yuan-Tzu Lan 1, 2 , Ming-Huang Chen 3, 6 , Wen-Liang Fang 2, 4 , Chih-Cheng Hsieh 5, 6 , Chien-Hsing Lin 7 , Fang-Yu Jhang 4 , Shung-Haur Yang 1, 2 , Jen-Kou Lin 1, 2 , Wei-Shone Chen 1, 2 , Jeng-Kai Jiang 1, 2 , Pei-Ching Lin 6, 8 , Shih-Ching Chang 1, 2 1 Division of Colon and Rectal Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan 2 Department of Surgery, National Yang-Ming University, Taipei, Taiwan 3 Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan 4 Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan 5 Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan 6 School of Medicine, National Yang-Ming University, Taipei, Taiwan 7 Genome Research Center, National Yang-Ming University, Taipei City, Taiwan 8 Department of Clinical Pathology, Yang-Ming Branch, Taipei City Hospital, Taipei, Taiwan Correspondence to: Pei-Ching Lin, email: b7901127@tmu.edu.tw Shih-Ching Chang, email: changsc@vghtpe.gov.tw Keywords: cfDNA, GI tract cancer, biomarker, carcinoembryonic antigen, response Received: September 08, 2016 Accepted: November 30, 2016 Published: December 07, 2016 ABSTRACT Background: Cell-free DNA (cfDNA) extracted from blood has become a clinically feasible biomarker in various types of cancer. However, the clinical significance of cfDNA in gastrointestinal (GI) tract cancer among Asian populations requires further investigation. Results: The median cfDNA copy number was highest in esophageal cancer, followed by colorectal cancer and gastric cancer, which were all significantly higher than those of healthy individuals. The cfDNA levels were higher in GI tract cancer, followed by those in carcinoma in situ and then healthy individuals ( P = 0.019). During the postoperative surveillance, the cfDNA level tended to be more sensitive than the carcinoembryonic antigen level in predicting recurrence. For recurrent gastric cancer, a persistently high cfDNA level and an increasing trend was observed after surgery. For stage IV colorectal cancer, dynamic changes in the cfDNA level were correlated with the responses to chemotherapy and surgery. Materials and Methods: Blood samples were collected from 95 healthy individuals and from 855 patients diagnosed with GI tract malignancy, including 98 with esophageal cancer, 428 with stomach cancer, 329 with colorectal cancer and 30 with carcinoma in situ . The copy numbers of extracted cfDNA were analyzed and compared among the different types of GI cancers. Conclusions: The cfDNA level can serve as a feasible biomarker for detecting tumors in GI tract cancer. The cfDNA level may play a role in predicting tumor responses to chemotherapy and surgery in colorectal cancer; tumor recurrence should be considered in gastric cancer with a persistently high cfDNA level after surgery.

To better understand the transmission and evolution of Mycobacterium tuberculosis (MTB) in Taiwan, six different MTB isolates (representatives of the Beijing ancient sublineage, Beijing modern sublineage, Haarlem, East-African Indian, T1, and Latin-American Mediterranean (LAM)) were characterized and their genomes were sequenced. Discriminating among large sequence polymorphisms (LSPs) that occur once versus those that occur repeatedly in a genomic region may help to elucidate the biological roles of LSPs and to identify the useful phylogenetic relationships. In contrast to our previous LSP-based phylogeny, the sequencing data allowed us to determine actual genetic distances and to define precisely the phylogenetic relationships between the main lineages of the MTB complex. Comparative genomics analyses revealed more nonsynonymous substitutions than synonymous changes in the coding sequences. Furthermore, MTB isolate M7, a LAM-3 clinical strain isolated from a patient of Taiwanese aboriginal origin, is closely related to F11 (LAM), an epidemic tuberculosis strain isolated in the Western Cape of South Africa. The PE/PPE protein family showed a higher dn/ds ratio compared to that for all protein-coding genes. Finally, we found Haarlem-3 and LAM-3 isolates to be circulating in the aboriginal community in Taiwan, suggesting that they may have originated with post-Columbus Europeans. Taken together, our results revealed an interesting association with historical migrations of different ethnic populations, thus providing a good model to explore the global evolution and spread of MTB.

With the progression of molecular techniques, the detection of circulating plasma DNA (cpDNA) is clinically feasible. However, the role of the cpDNA levels in gastric cancer is not well understood. This study assessed the mutational profile in primary tumors and clarified the clinical utility of quantitative and qualitative cpDNA alterations in 277 patients with advanced gastric cancer. The concentrations of cpDNA were measured by TaqMan qPCR, and 68 mutations in 8 genes were studied for cpDNA mutations. The median cpDNA concentrations in patients with stages I, II, and III gastric cancer were 3979, 3390 and 4278 copies/mL, respectively, and increased to 11,380 copies/mL in patients with Stage IV gastric cancer (p0.001). Among the 35 patients harboring cpDNA mutations, Stage IV patients (100%) were more likely to display high cpDNA levels than were Stage I (33.3%), II (75%) and III patients (66.7%) (p = 0.037). Patients displaying high cpDNA levels were more likely to experience peritoneal recurrence and exhibited significantly lower 5-year overall survival rates (39.2% vs. 45.8%, p = 0.039) than did patients displaying low cpDNA levels. Only for late stage (Stages III or IV) gastric cancer, patients harboring cpDNA mutations were more likely to experience vascular invasion (20% vs. 2.4%, p = 0.036) and exhibited a lower 5-year overall survival rate than did those lacking cpDNA mutations (5.6% vs. 31.5%, p = 0.028). High cpDNA levels are associated with peritoneal recurrence and poor prognosis in patients with advanced gastric cancer; harboring cpDNA mutations is associated with poor prognosis among patients with late stage gastric cancer.

e15591Background: Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of microsatellite instability (MSI) at tetranucleotide repeats and has emerged as a...