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Purpose: The goals of this study were to investigate the clinical activity, safety, and biomarkers of dacomitinib, an irreversible tyrosine kinase inhibitor of EGFR, HER2, and HER4, in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M-SCCHN).Experimental Design: Patients were eligible if the diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, and were treated with dacomitinib 45 mg/day. The primary endpoint was objective response rate by RECISTv1.1. Exploratory analysis included the characterization of somatic mutation, gene copy number, gene expression, p16INK4A expression by IHC, and investigation of their relationship with clinical outcomes.Results: Forty-eight patients were evaluable for efficacy and toxicity. Ten patients (20.8%) had partial responses and 31 patients (65%) had stable diseases. The median progression-free survival (PFS) and overall survival (OS) were 3.9 months [95% confidence interval (CI), 2.9–5.0] and 6.6 months (95% CI, 5.4–10.3). Adverse events were mostly grade 1–2. Mutations in the PI3K pathway (PIK3CA, PTEN) and high expression of inflammatory cytokines (IL6, IL8, IL1A, IL1B, IL4, and TNF) were significantly associated with shorter PFS (2.9 vs. 4.9 months without mutations, P = 0.013; 2.8 vs. 9.9 months with low expression, P = 0.004). Those harboring PI3K pathway mutations or high inflammatory cytokine expression had shorter median OS (6.1 vs. 12.5 months lacking PI3K pathway mutations and with low inflammatory cytokine expression, P = 0.005).Conclusions: Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and inflammatory cytokine expression may help identify which R/M-SCCHN patients are likely to gain benefit from dacomitinib. Clin Cancer Res; 21(3); 544–52. ©2014 AACR.

Supplementary Methods and Materials, Figures S1-S3, Tables S1-S5. 1. Supplementary Methods. page 2-4 -Gene set analysis for gene expression data -Immunohistochemistry and fluorescent in situ hybridization (FISH) -Enzyme-linked immunosorbent assay (ELISA) 2. Supplemental Figures. Figure S1: CONSORT diagram. page 5 Figure S2: Kaplan-Meier estimates of progression-free survival and overall survival according to p16 status. page 6 Figure S3: Analysis of fibroblast growth factor receptor 1 (FGFR1) gene amplification by fluorescent in situ hybridization. page 7 3. Supplemental Tables Table S1: Gene list and platforms for biomarker analysis. page 8-12 Table S2: Loci information of somatic mutations. page 13 Table S3: Gene expression profile in 230 cancer genes. page 14-20 Table S4: Gene set enrichment analysis. page 21-22 Table S5: Copy number aberrations in 86 cancer genes. page 23

Background Low-pass sequencing (LPS) has been extensively investigated for applicability to various genetic studies due to its advantages over genotype array data including cost-effectiveness. Predicting the risk of complex diseases such as Parkinson’s disease (PD) using polygenic risk score (PRS) based on the genetic variations has shown decent prediction accuracy. Although ultra-LPS has been shown to be effective in PRS calculation, array data has been favored to the majority of PRS analysis, especially for PD. Results Using eight high-coverage WGS, we assessed imputation approaches for downsampled LPS data ranging from 0.5 × to 7.0 × . We demonstrated that uncertain genotype calls of LPS diminished imputation accuracy, and an imputation approach using genotype likelihoods was plausible for LPS. Additionally, comparing imputation accuracies between LPS and simulated array illustrated that LPS had higher accuracies particularly at rare frequencies. To evaluate ultra-low coverage data in PRS calculation for PD, we prepared low-coverage WGS and genotype array of 87 PD cases and 101 controls. Genotype imputation of array and downsampled LPS were conducted using a population-specific reference panel, and we calculated risk scores based on the PD-associated SNPs from an East Asian meta-GWAS. The PRS models discriminated cases and controls as previously reported when both LPS and genotype array were used. Also strong correlations in PRS models for PD between LPS and genotype array were discovered. Conclusions Overall, this study highlights the potentials of LPS under 1.0 × followed by genotype imputation in PRS calculation and suggests LPS as attractive alternatives to genotype array in the area of precision medicine for PD.

Primary glioblastoma develops de novo without clinical or histological evidence of a low-grade precursor lesion, whereas secondary glioblastoma develops from a low-grade glioma. The present report describes an extraordinary case of IDH-wildtype secondary glioblastoma arising in IDH-mutant diffuse astrocytoma. A 31-year-old female had a surgical history of IDH-mutant diffuse astrocytoma on the left frontal lobe six years before. Magnetic resonance imaging revealed new infiltrative lesions in the left frontal lobe adjacent to the previous lesion. The patient underwent tumourectomy, and the new infiltrative lesion was diagnosed as glioblastoma. Interestingly, the IDH-1 (p.Arg132His) mutation was found in diffuse astrocytoma but not in glioblastoma based on next generation sequencing. ATRX (p.Gln1670Ter) and TP53 (p.His193Arg) mutations were found in both lesions. Additionally, the PTEN (p.His296Pro) mutation was identified only in glioblastoma. A well-accepted hypothesis is that the IDH mutation initiates in glial progenitor cells and causes secondary glioblastoma harboring the IDH mutation to develop from low grade glioma with IDH mutation. However, this case showed that the other genetic mutations can be initiated before the IDH mutation in glioma oncogenesis. Contrary to the previous hypothesis, this is the first case of IDH-wildtype secondary glioblastoma arising in IDH-mutant diffuse astrocytoma.

We evaluated the prognostic implications of the circulating tumor cell (CTC) count in non-metastatic, HER2-negative breast cancer patients who failed to achieve pathologic complete response (pCR) after neoadjuvant chemotherapy (NCT). A total of 173, non-metastatic breast cancer patients treated with NCT were prospectively enrolled. CTCs were obtained from blood drawn pre-NCT and post-NCT using a SMART BIOPSY SYSTEM isolation kit (Cytogen Inc., Seoul, Korea) with immunofluorescence staining. Excluding 26 HER2-positive patients, Relapse-free survival (RFS) and overall survival (OS) related to the CTC count and the association of the CTC count with the treatment response to given therapy were analyzed in 147 HER2-negative patients. Among 147 HER2-negative patients, 28 relapses (19.0%) and 13 deaths (8.8%, all breast cancer-specific) were observed during a median follow-up of 37.3 months. One hundred and seven patients (72.8%) were hormone receptor-positive, and 40 patients (27.2%) had triple-negative breast cancer (TNBC). One or more CTCs were identified in 88 of the 147 patients (59.9%) before NCT and 77 of the 134 patients (52.4%) after NCT. In the entire HER2-negative patient cohort, the initial nodal status was the most significant factor influencing RFS and OS. In TNBC, 11 patients (27.5%) achieved pCR and patients that failed to achieve pCR with ≥ 5 CTCs after NCT, showed worse RFS (HR, 10.66; 95% CI, 1.80–63.07; p = 0.009) and OS (HR, 14.00; 95% CI, 1.26–155.53; p = 0.032). The patients with residual tumor and a high number of the CTCs after NCT displayed the worse outcome. These findings could provide justification to launch a future, well designed trial with longer follow-up data to obtain regulatory approval for clinical use of the assay, especially for the ER-positive, HER2-negative breast cancer subset.

Accumulating evidence reveals the association between the risk of never-smoker lung cancer and family history of cancer. However, the clinicogenomic effect of family history of cancer in never-smoker lung cancer remains unknown.We screened 3,241 lung cancer patients who (a) underwent curative resection at National Cancer Center (Goyang, Korea) between 2001-2014, and (b) completed a pre-designed interview about family/smoking history at the time of diagnosis and identified 604 female never smoker lung adenocarcinoma. A positive family history of cancer [categorized as pulmonary cancer (FH-PC) or non-pulmonary cancer (FH-NPC)] was defined as a self-reported history of cancer in first-degree relatives. Survival data were followed up until January 2017. Multiplexed targeted next-generation sequencing was performed for genetic profiling.Of 604 patients, 29.1% (n = 176) had a FH, including 132 (21.9%) with FH-NPC and 44 (7.3%) with FH-PC. Patients with the FH-NPC had a higher proportion of young patients (≤45 years) than those without the FH-NPC (FH-NPC, FH-PC, and no FH; 13.6%, 2.3%, and 8.2%, respectively; P = 0.032). Patients with the FH-NPC had an increased risk of recurrence (hazard ratio [HR]: 1.90; 95% confidence interval [CI]: 1.40-2.56; P0.001) and death (HR: 1.67; 95% CI: 1.18-2.37; P=0.004). In contrast, the FH-PC had no prognostic effect on recurrence (HR: 1.23; 95% CI: 0.71-2.15; P = 0.456) and death (HR: 0.93; 95% CI: 0.45-1.91; P=0.838). Among three driver oncogene alterations, EGFR mutation was significantly associated with the FH-PC (53.8%, 84.1%, and 65.8%, respectively; P = 0.016), ALK/ROS1/RET fusions was significantly associated with the FH-NPC (13.7%, 0.0%, and 5.0%, respectively; P = 0.004), but KRAS mutation was not associated with any type of the FH (13.8% vs. 6.0% vs. 7.8%, respectively; P = 0.288).The type of family history of cancer was associated with distinct clinocogenomic subtypes and prognosis of never-smoker lung adenocarcinoma.

Extreme responders to anticancer therapy are rare among advanced breast cancer patients. Researchers, however, have yet to investigate treatment responses therein on the whole exome level. We performed whole exome analysis to characterize the genomic landscape of extreme responders among metastatic breast cancer patients. Clinical samples were obtained from breast cancer patients who showed exceptional responses to anti-HER2 therapy or hormonal therapy and from those who did not. Matched breast tumor tissue (somatic DNA) and blood samples (germline DNA) were collected from a total of 30 responders and 15 non-responders. Whole exome sequencing using Illumina HiSeq2500 was performed for all 45 patients (90 samples). Somatic single nucleotide variants (SNVs), indels, and copy number variants (CNVs) were identified for the genomes of each patient. Group-specific somatic variants and mutational burden were statistically analyzed. Sequencing of cancer exomes for all patients revealed 1839 somatic SNVs (1661 missense, 120 nonsense, 43 splice-site, 15 start/stop-lost) and 368 insertions/deletions (273 frameshift, 95 in-frame), with a median of 0.7 mutations per megabase (range, 0.08 to 4.2 mutations per megabase). Responders harbored a significantly lower nonsynonymous mutational burden (median, 26 vs. 59, P = 0.02) and fewer CNVs (median 13.6 vs. 97.7, P = 0.05) than non-responders. Multivariate analyses of factors influencing progression-free survival showed that a high mutational burden and visceral metastases were significantly related with disease progression. Extreme responders to treatment for metastatic breast cancer are characterized by fewer nonsynonymous mutations and CNVs., Lim, Kim and Jung et al. report a whole-exome sequence analysis of 30 Korean patients showing extreme treatment responses for metastatic breast cancer. They find that compared to non-responders, extreme responders have fewer nonsynonymous mutations and copy number variants.

We evaluated the prognostic implications of the circulating tumor cell (CTC) count in non-metastatic, HER2-negative breast cancer patients who failed to achieve pathologic complete response (pCR) after neoadjuvant chemotherapy (NCT). A total of 173, non-metastatic breast cancer patients treated with NCT were prospectively enrolled. CTCs were obtained from blood drawn pre-NCT and post-NCT using a SMART BIOPSY SYSTEM isolation kit (Cytogen Inc., Seoul, Korea) with immunofluorescence staining. Excluding 26 HER2-positive patients, Relapse-free survival (RFS) and overall survival (OS) related to the CTC count and the association of the CTC count with the treatment response to given therapy were analyzed in 147 HER2-negative patients. Among 147 HER2-negative patients, 28 relapses (19.0%) and 13 deaths (8.8%, all breast cancer-specific) were observed during a median follow-up of 37.3 months. One hundred and seven patients (72.8%) were hormone receptor-positive, and 40 patients (27.2%) had triple-negative breast cancer (TNBC). One or more CTCs were identified in 88 of the 147 patients (59.9%) before NCT and 77 of the 134 patients (52.4%) after NCT. In the entire HER2-negative patient cohort, the initial nodal status was the most significant factor influencing RFS and OS. In TNBC, 11 patients (27.5%) achieved pCR and patients that failed to achieve pCR with ≥ 5 CTCs after NCT, showed worse RFS (HR, 10.66; 95% CI, 1.80-63.07; p = 0.009) and OS (HR, 14.00; 95% CI, 1.26-155.53; p = 0.032). The patients with residual tumor and a high number of the CTCs after NCT displayed the worse outcome. These findings could provide justification to launch a future, well designed trial with longer follow-up data to obtain regulatory approval for clinical use of the assay, especially for the ER-positive, HER2-negative breast cancer subset.

The association between cancer and microbiome dysbiosis across anatomically related multiple body sites has not been comprehensively investigated. The purpose of our study is to profile microbial diversity and composition through the various gastrointestinal environment of gastric cancer (GC). We performed V3-V4 16S rRNA gene sequencing analysis for matched samples of gastric tumor, normal gastric mucosa, gastric juice and stool from 30 GC patients. Amplicon sequence variant (ASV) profile was compared among the four body sites at genus level. In this study, we found that mean alpha diversity was lowest in normal gastric mucosa and stool exhibited the largest amount of alpha diversity compared with others. Beta-diversity analysis showed significant differences in microbiota composition for each sample. The microbiome dysbiosis was significantly independent in gastrointestinal environment of gastric cancer. Helicobacter abundance in tumor tissue was significantly lower than in matched normal tissue and gastric juice while the trend was opposite for Lactobacillus. Additionally, the level of Helicobacter was considerably lower in patients with lymphatic invasion. The bacterial community that significantly correlated with tumor samples compared to normal mucosa, gastric juice, and stool were 49, 27, and 11 genus, respectively. Lactobacillus and Delftia had higher abundance and Rothia and Collnsella had lower abundance in tumor tissue compare with normal mucosa. Especially, Delftia was seen only in the tumor tissue not normal gastric mucosa, gastric juice and stool. Pentose phosphate pathway was significantly enriched in tumor tissue and normal mucosa. There is a unique microbiome pattern through the various gastrointestinal environment of gastric cancer. Our analysis shows enriched Delftia abundance only in the tumor tissue except other sample type. Citation Format: Jieun Lee, Sunguk Shin, Seung-been Lee, Yieri Yoo, Sangjun Lee, So Hyun Kang, Eunju Lee, Young Suk Park, Sang-Hoon Ahn, Kyoung Un Park, Hyung-Ho Kim, Nak Jung Kwon, Yun-Suhk Suh. Microbiome profiling through the various gastrointestinal environment of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6135.

Mutations in the EGFR gene downstream signaling pathways may cause receptor-independent pathway activation, making tumors unresponsive to EGFR inhibitors. However, the clinical significance of RAS, PIK3CA or PTEN mutations in NSCLC is unclear. In this study, patients who were initially diagnosed with NSCLC or experienced recurrence after surgical resection were enrolled, and blood samples was collected. Ultra-deep sequencing analysis of cfDNA using Ion AmpliSeq Cancer Hotspot Panel v2 with Proton platforms was conducted. RAS/PIK3CA/PTEN mutations were frequently detected in cfDNA in stage IV NSCLC (58.1%), and a high proportion of the patients (47.8%) with mutations had bone metastases at diagnosis. The frequency of RAS/PIK3CA/PTEN mutations in patients with activating EGFR mutation was 61.7%. The median PFS for EGFR-TKIs was 15.1 months in patients without RAS/PIK3CA/PTEN mutations, and 19.9 months in patients with mutations (p = 0.549). For patients with activating EGFR mutations, the overall survival was longer in patients without RAS/PIK3CA/PTEN mutations (53.8 months vs. 27.4 months). For the multivariate analysis, RAS/PIK3CA/PTEN mutations were independent predictors of poor prognosis in patients with activating EGFR mutations. In conclusion, RAS, PIK3CA and PTEN mutations do not hamper EGFR-TKI treatment outcome, however, they predict a poor OS when activating EGFR mutations coexist.

Novel coronavirus (SARS-CoV-2) is found to cause a large outbreak started from Wuhan since December 2019 in China and SARS-CoV-2 infections have been reported with epidemiological linkage to China in 25 countries until now. We isolated SARS-CoV-2 from the oropharyngeal sample obtained from the patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea. Cytopathic effects of SARS-CoV-2 in the Vero cell cultures were confluent 3 days after the first blind passage of the sample. Coronavirus was confirmed with spherical particle having a fringe reminiscent of crown on transmission electron microscopy. Phylogenetic analyses of whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan.

// Jae Sook Sung 1 , Hyon Yong Chong 2 , Nak-Jung Kwon 2 , Hae Mi Kim 2 , Jong Won Lee 1 , Boyeon Kim 1 , Saet Byeol Lee 1 , Chang Won Park 2 , Jung Yoon Choi 3 , Won Jin Chang 3 , Yoon Ji Choi 3 , Sung Yong Lee 4 , Eun Joo Kang 4 , Kyong Hwa Park 1, 3 and Yeul Hong Kim 1, 3 1 Cancer Research Institute, Korea University, Seoul, Republic of Korea 2 Macrogen, Seoul, Republic of Korea 3 Division of Oncology/Hematology, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Republic of Korea 4 Department of Internal Medicine, Guro Hospital, Korea University, Seoul, Republic of Korea Correspondence to: Yeul Hong Kim, email: yhk0215@korea.ac.kr Keywords: non-small-cell lung carcinoma; multiplex polymerase chain reaction; missense mutation; cell-free DNA; molecular targeted therapy Received: June 01, 2017 Accepted: October 17, 2017 Published: November 15, 2017 ABSTRACT Highly sensitive genotyping assays can detect mutations in cell-free DNA (cfDNA) from cancer patients, reflecting the biology of each patient’s cancer. Because circulating tumor DNA comprises a small, variable fraction of DNA circulating in the blood, sensitive parallel multiplexing tests are required to determine mutation profiles. We prospectively examined the clinical utility of ultra-deep sequencing analysis of cfDNA from 126 non-small cell lung cancer (NSCLC) patients using the Ion AmpliSeq Cancer Hotspot Panel v2 (ICP) and validated these findings with droplet digital polymerase chain reaction (ddPCR). ICP results were compared with tumor tissue genotyping (TTG) results and clinical outcomes. A total of 853 variants were detected, with a median of four variants per patient. Overall concordance of ICP and TTG analyses was 90% for EGFR exon 19 deletion and 88% for the L858R mutation. Of 34 patients with a well-defined EGFR activating mutation defined based on the results of ICP and TTG, 31 (81.6%) showed long-term disease control with EGFR TKI treatment. Of 56 patients treated with an EGFR tyrosine kinase inhibitor (TKI), the presence of the de novo T790M mutation was confirmed in 28 (50%). Presence of this de novo mutation did not have a negative effect on EGFR TKI treatment. Ultra-deep sequencing analysis of cfDNA using ICP combined with confirmatory ddPCR was effective at defining driver genetic changes in NSCLC patients. Comprehensive analysis of tumor DNA and cfDNA can increase the specificity of molecular diagnosis, which could translate into tailored treatment.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

The increase in genetic alterations targeted by specific chemotherapy in lung cancer has led to the need for universal use of more comprehensive genetic testing, which has highlighted the development of a lung cancer diagnostic panel using next-generation sequencing. We developed a hybridization capture-based massively parallel sequencing assay named Friendly, Integrated, Research-based, Smart and Trustworthy (FIRST)-lung cancer panel (LCP), and evaluated its performance. FIRST-LCP comprises 64 lung cancer-related genes to test for various kinds of genetic alterations including single nucleotide variations (SNVs), insertions and deletions (indels), copy number variations (CNVs), and structural variations. To assess the performance of FIRST-LCP, we compiled test sets using HapMap samples or tumor cell lines with disclosed genetic information, and also tested our clinical lung cancer samples whose genetic alterations were known by conventional methods. FIRST-LCP accomplished high sensitivity (99.4%) and specificity (100%) of the detection of SNVs. High precision was also achieved, with intra- or inter-run concordance rate of 0.99, respectively. FIRST-LCP detected indels and CNVs close to the expected allele frequency and magnitude, respectively. Tests with samples from lung cancer patients also identified all SNVs, indels and fusions. Based on the current state of the art, continuous application of the panel design and analysis pipeline following up-to-date knowledge could ensure precision medicine for lung cancer patients.

// Sun Min Lim 1,2,* , Hyung Soon Park 3,* , Sangwoo Kim 4 , Sora Kim 4 , Siraj M. Ali 5 , Joel R. Greenbowe 5 , In Seok Yang 4 , Nak-Jung Kwon 6 , Jae Lyun Lee 7 , Min-Hee Ryu 7 , Jin-Hee Ahn 7 , Jeeyun Lee 8 , Min Goo Lee 3 , Hyo Song Kim 1 , Hyunki Kim 9 , Hye Ryun Kim 1 , Yong Wha Moon 1,2 , Hyun Cheol Chung 1 , Joo-Hang Kim 2 , Yoon-Koo Kang 7 and Byoung Chul Cho 1 1 Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea 2 Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea 3 Department of Pharmacology and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 4 Severance Biomedical Science Institute and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 5 Foundation Medicine Inc, Cambridge, MA, USA 6 MacroGen Inc., Seoul, Korea 7 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 8 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 9 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Yoon-Koo Kang, email: // Byoung Chul Cho, email: // Keywords : everolimus, NF1, TSC1, mTOR, next-generation sequencing Received : November 27, 2015 Accepted : January 25, 2016 Published : February 07, 2016 Abstract Background: Given the modest responses to everolimus, a mTOR inhibitor, in multiple tumor types, there is a pressing need to identify predictive biomarkers for this drug. Using targeted ultra-deep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. Results: We collected formalin-fixed paraffin-embedded tumor/normal pairs from 39 patients (22 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 2 head and neck cancer, and 7 sarcomas). Ion AmpliSeq TM Comprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Tumors were sequenced to a median coverage of 552x. Cancer genomes are characterized by 219 somatic single-nucleotide variants (181 missense, 9 nonsense, 7 splice-site) and 22 frameshift insertions/deletions, with a median of 2.1 mutations per Mb (0 to 12.4 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 10 of 22 (45%) patients with clinical benefit and these include MTOR, TSC1, TSC2, NF1 , PIK3CA and PIK3CG mutations. Recurrently mutated genes in chromatin remodeling genes ( BAP1; n = 2, 12%) and receptor tyrosine kinase signaling ( FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. Conclusions: Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors.

// Hyo Song Kim 1,* , Sung-Moo Kim 2,* , Hyunki Kim 3 , Kyoung-Ho Pyo 2 , Jong-Mu Sun 4 , Myung-Ju Ahn 4 , Keunchil Park 4 , Bhumsuk Keam 5 , Nak-Jung Kwon 6 , Hwan Jung Yun 7 , Hoon-Gu Kim 8 , Ik-Joo Chung 9 , Jong Seok Lee 10 , Kyung Hee Lee 11 , Dae Joon Kim 12 , Chang-Geol Lee 13 , Jin Hur 14 , Hyunsoo Chung 15 , Jun Chul Park 15 , Sung Kwan Shin 15 , Sang Kil Lee 15 , Hye Ryun Kim 1 , Yong Wha Moon 1 , Yong Chan Lee 15 , Joo Hang Kim 1 , Soonmyung Paik 16,17 and Byoung Chul Cho 1 1 Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea 2 Yonsei Cancer Research Institute, JE-UK Laboratory of Molecular Cancer Therapeutics, Seoul, Korea 3 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea 4 Department of Hematology-Oncology, Samsung Medical Center, Seoul, Korea 5 Department of Hematology-Oncology, Seoul National University Hospital, Seoul, Korea 6 Macrogen Inc., Seoul, Korea 7 Division of Hemato-Oncology, Chungnam National University Hospital, Daejeon, Korea 8 Division of Hematology-Oncology, Department of Internal Medicine, Gyeongnam Regional Cancer Center, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea 9 Department of Hematology-Oncology, Chonnam National University Hwasun Hospital, Hwasun, South Korea 10 Department of Hematology-Oncology, Seoul National University Bundang Hospital, Seongnam, Korea 11 Department of Hematology-Oncology, Yeungnam University Medical Center, Daegu, South Korea 12 Department of Thoracic and Cardiovascular Surgery, Yonsei University College of Medicine, Seoul, Korea 13 Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Korea 14 Department of Radiology, Yonsei University College of Medicine, Seoul, Korea 15 Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea 16 Division of Pathology NSABP, Pittsburgh, PA, USA 17 Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea * These authors have contributed equally to this work as co-first authors Correspondence to: Byoung Chul Cho, email: // Keywords : epidermal growth factor receptor, tyrosine kinase inhibitor, esophageal squamous cell carcinoma, biomarker Received : June 25, 2015 Accepted : September 23, 2015 Published : October 09, 2015 Abstract The purpose of this study was to investigate the clinical activity, safety and predictive biomarkers of dacomitinib, an irreversible pan-HER inhibitor, in patients with recurrent or metastatic esophageal squamous cell carcinoma (R/M-ESCC). Patients, whose diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, were treated with dacomitinib 45mg/day. The primary endpoint was objective response rate by RECISTv 1.1. Predictive biomarker analyses included the characterization of somatic mutations and gene expression using the Ion Torrent AmpliSeq Cancer Hotspot Panel and Nanostring nCounter, and investigation of their relationship with clinical outcomes. Of the 48 evaluable patients, 6 (12.5%) achieved partial responses and 29 (60.4%) had stable disease. The median response duration was 7.1 months. The median progression free survival (PFS) and overall survival (OS) was 3.3 months (95% CI, 2.4-4.3 months) and 6.4 months (95% CI, 4.4-8.4 months). Adverse events were mostly grade 1-2. Gene set enrichment analysis revealed that ERBB signaling pathway is significantly enriched in patients with PFS ≥4 months ( n = 12) than PFS < 4 months ( n = 21) ( p < 0.001). Upregulation of ERBB signaling pathway was significantly associated with longer PFS (5.0 vs. 2.9 months, P = 0.016) and OS (10.0 vs. 4.8 months, P = 0.022). The most frequent mutations were TP53 (61%) followed by CDKN2A (8%), MLH1 (8%), FLT3 (8%) and EGFR (8%). Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-ESCC. Screening of ERBB pathway-related gene expression profiles may help identify patients who are most likely benefit from dacomitinib.

Purpose: The goals of this study were to investigate the clinical activity, safety, and biomarkers of dacomitinib, an irreversible tyrosine kinase inhibitor of EGFR, HER2, and HER4, in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M-SCCHN). Experimental Design: Patients were eligible if the diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, and were treated with dacomitinib 45 mg/day. The primary endpoint was objective response rate by RECISTv1.1. Exploratory analysis included the characterization of somatic mutation, gene copy number, gene expression, p16INK4A expression by IHC, and investigation of their relationship with clinical outcomes. Results: Forty-eight patients were evaluable for efficacy and toxicity. Ten patients (20.8%) had partial responses and 31 patients (65%) had stable diseases. The median progression-free survival (PFS) and overall survival (OS) were 3.9 months [95% confidence interval (CI), 2.9–5.0] and 6.6 months (95% CI, 5.4–10.3). Adverse events were mostly grade 1–2. Mutations in the PI3K pathway (PIK3CA, PTEN) and high expression of inflammatory cytokines (IL6, IL8, IL1A, IL1B, IL4, and TNF) were significantly associated with shorter PFS (2.9 vs. 4.9 months without mutations, P = 0.013; 2.8 vs. 9.9 months with low expression, P = 0.004). Those harboring PI3K pathway mutations or high inflammatory cytokine expression had shorter median OS (6.1 vs. 12.5 months lacking PI3K pathway mutations and with low inflammatory cytokine expression, P = 0.005). Conclusions: Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and inflammatory cytokine expression may help identify which R/M-SCCHN patients are likely to gain benefit from dacomitinib. Clin Cancer Res; 21(3); 544–52. ©2014 AACR.

Highly sensitive genotyping assays can detect mutations in cell-free DNA (cfDNA) from cancer patients, reflecting the biology of each patient’s cancer. Because circulating tumor DNA comprises a small, variable fraction of DNA circulating in the blood, sensitive parallel multiplexing tests are required to determine mutation profiles. We prospectively examined the clinical utility of ultra-deep sequencing analysis of cfDNA from 126 non-small cell lung cancer (NSCLC) patients using the Ion AmpliSeq Cancer Hotspot Panel v2 (ICP) and validated these findings with droplet digital polymerase chain reaction (ddPCR). ICP results were compared with tumor tissue genotyping (TTG) results and clinical outcomes. A total of 853 variants were detected, with a median of four variants per patient. Overall concordance of ICP and TTG analyses was 90% for EGFR exon 19 deletion and 88% for the L858R mutation. Of 34 patients with a well-defined EGFR activating mutation defined based on the results of ICP and TTG, 31 (81.6%) showed long-term disease control with EGFR TKI treatment. Of 56 patients treated with an EGFR tyrosine kinase inhibitor (TKI), the presence of the de novo T790M mutation was confirmed in 28 (50%). Presence of this de novo mutation did not have a negative effect on EGFR TKI treatment. Ultra-deep sequencing analysis of cfDNA using ICP combined with confirmatory ddPCR was effective at defining driver genetic changes in NSCLC patients. Comprehensive analysis of tumor DNA and cfDNA can increase the specificity of molecular diagnosis, which could translate into tailored treatment.

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (−) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (−) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.

Asexual development (conidiation) in the filamentous fungus Aspergillus nidulans is governed by orchestrated gene expression. The three key negative regulators of conidiation SfgA, VosA, and NsdD act at different control point in the developmental genetic cascade. Here, we have revealed that NsdD is a key repressor affecting the quantity of asexual spores in Aspergillus. Moreover, nullifying both nsdD and vosA results in abundant formation of the development specific structure conidiophores even at 12 h of liquid culture, and near constitutive activation of conidiation, indicating that acquisition of developmental competence involves the removal of negative regulation exerted by both NsdD and VosA. NsdD’s role in repressing conidiation is conserved in other aspergilli, as deleting nsdD causes enhanced and precocious activation of conidiation in Aspergillus fumigatus or Aspergillus flavus. In vivo NsdD-DNA interaction analyses identify three NsdD binding regions in the promoter of the essential activator of conidiation brlA, indicating a direct repressive role of NsdD in conidiation. Importantly, loss of flbC or flbD encoding upstream activators of brlA in the absence of nsdD results in delayed activation of brlA, suggesting distinct positive roles of FlbC and FlbD in conidiation. A genetic model depicting regulation of conidiation in A. nidulans is presented.

Mitochondria play key roles in cellular energy generation and lifespan of most eukaryotes. To understand the functions of four nuclear-encoded genes predicted to be related to the maintenance of mitochondrial morphology and function in Aspergillus nidulans, systematic characterization was carried out. The deletion and overexpression mutants of aodA, dnmA, mnSOD and pimA encoding alternative oxidase, dynamin related protein, manganese superoxide dismutase and Lon protease, respectively, were generated and examined for their growth, stress tolerances, respiration, autolysis, cell death, sterigmatocystin production, hyphal morphology and size and mitochondrial superoxide production as well as development. Overall, genetic manipulation of these genes had less effect on cellular physiology and ageing in A. nidulans than that of their homologs in another fungus Podospora anserina with a well-characterized senescence. The observed interspecial phenotypic differences can be explained by the dissimilar intrinsic stabilities of the mitochondrial genomes in A. nidulans and P. anserina. Furthermore, the marginally altered phenotypes observed in A. nidulans mutants indicate the presence of effective compensatory mechanisms for the complex networks of mitochondrial defense and quality control. Importantly, these findings can be useful for developing novel platforms for heterologous protein production, or on new biocontrol and bioremediation technologies based on Aspergillus species.

During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA , which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus . In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans ) or near (for A. fumigatus ) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (Af ricA ) restores growth and conidiation in the ΔAn ricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus . A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA , flbA , rgsB , or rgsC , restored vegetative growth and conidiation in ΔAn ricA . Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro . Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAn ricA , indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans . Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the Δ ricA mutant. In addition, mutants overexpressing fluG or brlA (OE fluG or OE brlA ) failed to restore development in the ΔAn ricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans .

Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG-BrlA asexual developmental signaling and modulated by PacC-dependent pH-responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The β-lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine-tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two-component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca(2+) -homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review.

Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C(2) H(2) zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans.

The roles of the Gbetagamma subunits of the opportunistic human pathogen Aspergillus fumigatus were investigated. The predicted AfuSfaD (Gbeta) protein consists of 353 amino acids and shows 94-98% similarity with other Aspergillus Gbeta subunits. AfuGpgA consists of 90 amino acids showing 95-98% identity with other fungal G-protein gamma subunits. The deletion (Delta) of AfusfaD or AfugpgA resulted in severe impairment in vegetative growth, conidial germination and conidial trehalose breakdown. While the total number of conidia produced by DeltaAfusfaD and DeltaAfugpgA strains on solid medium was only about 1% of wild type, the growth-adjusted conidiation levels were twofold higher than those of wild type. Enhanced formation of conidiophores and elevated AfubrlA mRNA levels were observable in DeltaAfusfaD or DeltaAfugpgA strains in liquid submerged culture. Moreover, overexpression of AfusfaD or AfugpgA caused reduced levels of submerged culture conidiation, indicating that Gbetagamma is involved in negative regulation of conidiation. Gliotoxin and other metabolites were not detected in the chloroform extracts of DeltaAfusfaD and DeltaAfugpgA culture filtrates. Northern blot analyses revealed that, while AfulaeA mRNA levels unchanged, accumulation of gliZ mRNA was delayed by DeltaAfusfaD or DeltaAfugpgA. A model summarizing the roles of AfusfaD and AfugpgA in A. fumigatus is presented.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Although numerous effective therapies have improved the survival rate of patients with breast cancer, a number of patients present with tumor recurrence and metastasis. A liquid biopsy of circulating tumor cells (CTC) is a non-invasive method to obtain tumor cells and may be used as substitute for a tumor tissue biopsy. The present study focuses on determining whether CTC culture is an optimal method of obtaining sufficient amounts of CTCs for molecular analysis. The current study demonstrates a method of isolating and culturing CTCs from patients with breast cancer and the construction of a molecular profile of cultured cells using the Ion AmpliSeq Cancer Gene Panel V2. Gene mutations that were observed in cultured CTCs were compared with those observed in primary tumor tissues. CTCs were isolated and cultured from the blood of six patients with breast cancer. Mutations from the Catalogue Of Somatic Mutation In Cancer (COSMIC) were detected in Platelet-Derived Growth Factor Receptor Alpha, MET (also known as Hepatocyte Growth Factor Receptor), Phosphatase and Tensin Homolog, Harvey Rat Sarcoma Viral Oncogene Homolog, SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily B Member 1, Cyclin Dependent Kinase Inhibitor 2A and MutL Homolog 1 genes in 5/6 samples. A comparison between mutations detected in cultured CTCs and mutations detected in primary tumor tissues demonstrated that a large number of mutations that were identified in CTCs were also detected in primary tumor tissues. The results from the current study describe a novel cell culture approach that may be used to obtain an optimal number of CTCs for molecular analysis. This novel approach is able to be used as a tool for liquid biopsy during breast cancer treatment.

Cell free DNA (cfDNA) present in the blood stream shows great potential as a useful cancer marker for molecular diagnosis and cancer progression monitoring. Especially, analyzing the cfDNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. However, there are still debates regarding clinically meaningful variant frequency to identify mutations in cfDNA, especially with ultra-deep sequencing. In this study, we examined the clinical utility of Ion AmpliSeq Cancer Hotspot Panel v2 (ICP; Ion Torrent) with Proton platforms. ICP, covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from lung cancer patients. The percentage of on target was 92% with mean depth of 22,868x. We identified aberrations of TP53 (72%), EGFR (43%), PTEN (26%), PIK3CA (26%), BRAF (16%), KRAS (14%), KIT (10%) and RET (10%) with the cut-off criteria of variant frequency >0.1% and p Citation Format: Jae Sook Sung, Jong Won Lee, Boyeon Kim, Saet Byeol Lee, Chang Won Park, Hae Mi Kim, Nak-Jung Kwon, Won Jin Jang, Yoon Ji Choi, Jung Yoon Choi, Eun Joo Kang, Kyung Hwa Park, Sung Yong Lee, Yeul Hong Kim. Clinical significance of low frequency EGFR and KRAS mutations of cell free DNA using Ion AmpliSeq Cancer Hotspot Panel in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5680. doi:10.1158/1538-7445.AM2017-5680

Smoking is the major risk factor for lung squamous cell carcinoma (SCC), although a small number of lung SCCs occurs in never-smokers. The purpose of this study was to compare 50 hotspot mutations of lung SCCs between never-smokers and smokers. We retrospectively reviewed the medical records of patients newly diagnosed with lung SCC between January 1, 2011 and December 31, 2013 in the Seoul National University Hospital. Formalin-fixed, paraffin-embedded tumor samples were used for analysis of hotspot mutations. Fifty cancer-related genes in never-smokers were compared to those in ever-smokers. Of 379 lung SCC patients, 19 (5.0%) were never-smokers. The median age of these 19 patients was 67 years (interquartile range 57–73 years), and 10 of these patients were women (52.5%). The incidence rates of stage I, II, III, and IV disease in this group were 26.4%, 5.3%, 31.6%, and 36.8%, respectively, and sequencing was performed successfully in 14 cases. In the 26 lung SCC tumor samples (12 from never-smokers and 14 from ever-smokers) sequenced using personal genome machine, the most common mutations were in TP53 (75.0%), RAS (66.7%), and STK11 (33.3%), but mutations were also found in EGFR, KIT, and PTEN. The distribution of hotspot mutations in never-smokers was similar to that in ever-smokers. There was no significant difference in overall survival between the 2 groups. The 50 hotspot mutations of lung SCC in never-smokers were similar to those of ever-smokers., Graphical Abstract

The removal of polycyclic aromatic hydrocarbons by soil washing using water is extremely difficult due to their intrinsic hydrophobic nature. In this study, the effective aqueous solubility enhancements of seven polycyclic aromatic hydrocarbons by chemically modified hydroxypropyl rhizobial cyclic β-(1 → 2)-D-glucans and epichlorohydrin β-cyclodextrin dimer have been investigated for the first time. In the presence of hydroxypropyl cyclic β-(1 → 2)-D-glucans, the solubility of benzo[a]pyrene is increased up to 38 fold of its native solubility. The solubility of pyrene and phenanthrene dramatically increased up to 160 and 359. Coronene, chrysene, perylene, and fluoranthene also show an increase of 11, 23, 23, and 97 fold, respectively, of enhanced solubility by complexation with synthetic epichlorohydrin β-cyclodextrin dimer. The physicochemical properties of the complex are characterized by Fourier-transform infrared spectra and differential scanning calorimetry. Utilizing a scanning electron microscopy, the morphological structures of native benzo[a]pyrene, pyrene, phenanthrene, coronene, chrysene, perylene, fluoranthene and their complex with novel carbohydrate-solubilizers are studied. These results elucidate that polycyclic aromatic hydrocarbons are able to form an efficient complex with hydroxypropyl cyclic β-(1 → 2)-D-glucans and β-cyclodextrin dimer, suggesting the potential usage of chemically modified novel carbohydrate-solubilizers.

This chapter discusses our current understanding of how conidiation in Aspergillus is regulated, with emphasis on the model fungus A. nidulans. The three genes (brlA, abaA, and wetA) have been proposed to define a central regulatory pathway that acts in concert with other genes to control conidiation-specific gene expression and determine the order of gene activation during conidiophore development and spore maturation. Identification and characterization of the developmental activators greatly enhanced our understanding of the molecular mechanisms for upstream regulation of conidiation in Aspergillus. Mutational inactivation of flbB, flbC, flbD, or flbE results in a third, distinct class of developmental defects classified as delayed conidiation. Importantly, while mutational inactivation of any one of these G protein components restores conidiation of the ΔflbA mutant to a certain level, no mutation can bypass the need for FluG during conidiation. Although asexual sporulation is the most common reproductive mode of many filamentous fungi, little is known about the regulatory mechanisms controlling this process. VosA plays two principal roles: (i) coupling sporogenesis and trehalose biogenesis to complete spore maturation and (ii) exerting negative-feedback regulation of developmental specific genes by repressing the expression of brlA encoding the key activator of conidiation in Aspergillus. VosA and related velvet proteins are found to be crucial global regulators for fungal development and metabolism.

Aspergillus nidulans exhibited high γ-glutamyl transpeptidase (γGT) activity in both carbon-starved and carbon-limited cultures. Glucose repressed, but casein peptone increased γGT production. Null mutation of creA did not influence γGT formation, but the functional meaB was necessary for the γGT induction. Deletion of the AN10444 gene (ggtA) completely eliminated the γGT activity, and the mRNA levels of ggtA showed strong correlation with the observed γGT activities. While ggtA does not contain a canonical signal sequence, the γGT activity was detectable both in the fermentation broth and in the hyphae. Deletion of the ggtA gene did not prevent the depletion of glutathione observed in carbon-starved and carbon-limited cultures. Addition of casein peptone to carbon-starved cultures lowered the formation of reactive species (RS). Deletion of ggtA could hinder this decrease and resulted in elevated RS formation. This effect of γGT on redox homeostasis may explain the reduced cleistothecia formation of ΔggtA strains in surface cultures.

Non-small cell lung cancers (NSCLC) are characterized by a unique pattern of genetic driver mutations, and some of mutations may be used to predict prognosis and targeted treatment such as EGFR TKIs. Cell free (cfDNA) present in the blood stream shows much potential as a useful cancer maker for early diagnosis and cancer progression monitoring. Especially, analyzing the cfDNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. However, there are still no standardized methods to identify mutations in cfDNA. In this study, we examined the viability of PGM and Proton platforms. Ion AmpliSeq Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from NSCLC patients. The on target was 88% and mean depth was 643x using PGM platform. And, the on target was 92% and mean depth was 22,868x in Proton platform. To validate the results of two NGS platforms, we analyzed EGFR status by sanger sequencing in available 100 tumor tissues. EGFR mutations were identified in 34 (34%) by sanger sequencing. EGFR mutations were identified in 32 (25.6%) and 28 (22.4%) by PGM and Proton platform. Interestingly, out of 34 mutations of tumor tissue, EGFR mutations were matched to 11 and 26 in PGM and Proton platform, respectively. These results showed concordance of 76.5% between the tDNA (sanger sequencing) and cfDNA (Proton). In addition, KRAS (codon 12 and 13) mutations were 4 (3.2%) and 18 (14.4%), respectively. In our study, we demonstrated that Proton platform of high depth is a useful assay to identify somatic mutations of cfDNA in NSCLCs. [This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0066)] Citation Format: Jae Sook Sung, Jong Won Lee, Boyeon Kim, Saet Byeol Lee, Nak-Jung Kwon, Won-Chul Lee, Hae Mi Kim, Won Jin Jang, Yun Ji Choi, Kyung Hwa Park, Yeul Hong Kim. Comparison and evaluation of somatic mutation using PGM and proton platform in cell free DNA of non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3614.

The β-lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine-tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two-component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca(2+) -homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review.

Aims: To elucidate the roles of the β-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. Methods and Results: A β-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. Conclusions: The β-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall–degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. Significance and Impact of the Study: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.

Aims: Elucidation of the regulation of ChiB production in Aspergillus nidulans. Methods and Results: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. Conclusions: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. Significance and Impact of the Study: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans . Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (Δ flbA ) mutant autolyzed completely after a 3-day incubation, the Δ flbA Δ chiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the Δ flbA -induced autolysis. However, importantly, both Δ flbA and Δ flbA Δ chiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the Δ flbA and Δ flbA Δ chiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O -glycosyl compound hydrolase, β- N -acetylhexosaminidase (NagA), and myo -inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.

Capsicum annuum RING Zinc Finger Protein 1 (CaRZFP1) gene is a novel C3HC4-type RING zinc finger protein gene which was previously isolated from a cDNA library for hot pepper plants treated of heat-shock. The CaRZFP1 was inducible to diverse environmental stresses in hot pepper plants. We introduced the CaRZFP1 into the Wisconsin 38 cultivar of tobacco (Nicotiana tabacum) by Agrobacterium mediated transformation under the control of the CaMV 35S promoter. Expression of the transgene in the transformed tobacco plants was demonstrated by RNA blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T(1) lines showed that the transgene segregated in a Mendelian fashion. Transgenic tobacco lines that expressed the CaRZFP1 gene were compared with several different empty vector lines and they exhibited enhanced growth; they have larger primary root, more lateral root, larger hypocotyls and bigger leaf size, resulting in heavier fresh weight. Enhanced growth of transgenic lines accompanied with longer vegetative growth that resulted in bigger plants with higher number of leaves. Microarray analysis revealed the up-regulation of some growth related genes in the transgenic plants which were verified by specific oligomer RNA blot analyses. These results indicate that CaRZFP1 activates and up-regulates some growth related proteins and thereby effectively promoting plant growth.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans , light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

11 páginas, 8 figuras, 3 tablas -- PAGS nros. 38-48, The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation, The work carried out at UW-Madison was supported by Hatch (WIS04667) and National Science Foundation (MCB-0421863 and IOS-0640067) grants to J.H.Y. E.A.E thanks Ministerio de Educación y Ciencia for support through grant BFU2006-04185. U.U. thanks Ministerio de Educación y Ciencia for grant BFU2004-03499/BMC and UPV/EHU for grant GIU05/36. O.E received a doctoral grant from UPV/EHU, and A.G. was a contract researcher under the research program of Gipuzkoako Foru Aldundia/Diputación Foral de Gipuzkoa. The work was also supported by the Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany