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Supplementary Fig. S1. Schematic protocol of tissue collection in five colon cancers with liver metastasis. In each colon cancer patient, 2 to 5 tumor fragments each apart at least from 0.5 cm were collected from colon. In parallel, 2 to 6 separate metastatic nodules in the liver were collected from each patient.

Purpose: The intratumoral heterogeneity (ITH) and the evolution of genomic architectures associated with the development of distant metastases are not well understood in colorectal cancers.Experimental Design: We performed multiregion biopsies of primary and liver metastatic regions from five colorectal cancers with whole-exome sequencing and copy number profiling.Results: In addition to a substantial level of genetic ITH, multiregion genetic profiling identifies the subclonal mutational architecture, leading to the region-based or spatial categorization of somatic mutations and the inference of intratumoral evolutionary history of cancers. The universal mutations (those observed in all the regional biopsies) are enriched in known cancer genes such as APC and TP53 with distinct mutational spectra compared with biopsy- or region-specific mutations, suggesting that major operative mutational mechanisms and their selective pressures are not constant across the metastatic progression. The phylogenies inferred from genomic data show branching evolutionary patterns where some primary biopsies are often segregated with metastastic lesions. Our analyses also revealed that copy number changes such as the chromosomal gains of c-MYC and chromothripsis can be region specific and the potential source of genetic ITH.Conclusions: Our data show that the genetic ITH is prevalent in colorectal cancer serving as a potential driving force to generate metastasis-initiating clones and also as a means to infer the intratumoral evolutionary history of cancers. The paucity of recurrent metastasis-clonal events suggests that colorectal cancer distant metastases may not follow a uniform course of genomic evolution, which should be considered in the genetic diagnosis and the selection of therapeutic targets for the advanced colorectal cancer. Clin Cancer Res; 21(19); 4461–72. ©2015 AACR.

Supplementary Table 1. Clinicopathological and sequencing information of the cases and regional biopsies

Supplementary Table 2. The mutations observed at least once for the regional biopsies across five CRC genomes. Genomic coordinates are according to hg19. The sequencechanges (Ref and Alt as reference and mutant sequences, respectively) are shown with the corresponding functional categories as reported by ANNOVAR. The ANNOVARreports are shown in the separate column (Annovar description). The color schemes for SC (spatial categories) are as shown in Main Figure 1. The mutations in the biopsies of primary (black; P) and metastatic lesions (purple; M) are shown in the matrix.

Despite great advances in understanding of molecular pathogenesis and achievement of a high cure rate in medulloblastoma, recurrent medulloblastomas are still dismal. Additionally, misidentification of secondary malignancies due to histological ambiguity leads to misdiagnosis and eventually to inappropriate treatment. Nevertheless, the genomic characteristics of recurrent medulloblastomas are poorly understood, largely due to a lack of matched primary and recurrent tumor tissues. We performed a genomic analysis of recurrent tumors from 17 pediatric medulloblastoma patients. Whole transcriptome sequencing revealed that a subset of recurrent tumors initially diagnosed as locally recurrent medulloblastomas are secondary glioblastomas after radiotherapy, showing high similarity to the non-G-CIMP proneural subtype of glioblastoma. Further analysis, including whole exome sequencing, revealed missense mutations or complex gene fusion events in PDGFRA with augmented expression in the secondary glioblastomas after radiotherapy, implicating PDGFRA as a putative driver in the development of secondary glioblastomas after treatment exposure. This result provides insight into the possible application of PDGFRA-targeted therapy in these second malignancies. Furthermore, genomic alterations of TP53 including 17p loss or germline/somatic mutations were also found in most of the secondary glioblastomas after radiotherapy, indicating a crucial role of TP53 alteration in the process. On the other hand, analysis of recurrent medulloblastomas revealed that the most prevalent alterations are the loss of 17p region including TP53 and gain of 7q region containing EZH2 which already exist in primary tumors. The 7q gain events are frequently accompanied by high expression levels of EZH2 in both primary and recurrent medulloblastomas, which provides a clue to a new therapeutic target to prevent recurrence. Considering the fact that it is often challenging to differentiate between recurrent medulloblastomas and secondary glioblastomas after radiotherapy, our findings have major clinical implications both for correct diagnosis and for potential therapeutic interventions in these devastating diseases.

Despite advances in medulloblastoma treatment and research, recurrent medulloblastoma is still devastating. Additionally, misidentification of secondary malignancy due to histological ambiguity leads to misdiagnosis and eventual inappropriate treatment. Through deep-sequencing of RNA and whole-exome of recurrent tumors from 17 pediatric medulloblastomas, we found that 3 patients had radiation-induced glioblastoma rather than recurrent medulloblastomas. We also uncovered that PDGFRA is a putative driver in radiation-induced glioblastoma, which gives an insight into the possible application of PDGFRA-targeted therapy. The observation of frequent gain of 7q region containing EZH2 accompanied by its high expression both in primary and recurrent medulloblastomas provides a clue to new a therapeutic target to prevent recurrence.

Purpose: The intratumoral heterogeneity (ITH) and the evolution of genomic architectures associated with the development of distant metastases are not well understood in colorectal cancers. Experimental Design: We performed multiregion biopsies of primary and liver metastatic regions from five colorectal cancers with whole-exome sequencing and copy number profiling. Results: In addition to a substantial level of genetic ITH, multiregion genetic profiling identifies the subclonal mutational architecture, leading to the region-based or spatial categorization of somatic mutations and the inference of intratumoral evolutionary history of cancers. The universal mutations (those observed in all the regional biopsies) are enriched in known cancer genes such as APC and TP53 with distinct mutational spectra compared with biopsy- or region-specific mutations, suggesting that major operative mutational mechanisms and their selective pressures are not constant across the metastatic progression. The phylogenies inferred from genomic data show branching evolutionary patterns where some primary biopsies are often segregated with metastastic lesions. Our analyses also revealed that copy number changes such as the chromosomal gains of c-MYC and chromothripsis can be region specific and the potential source of genetic ITH. Conclusions: Our data show that the genetic ITH is prevalent in colorectal cancer serving as a potential driving force to generate metastasis-initiating clones and also as a means to infer the intratumoral evolutionary history of cancers. The paucity of recurrent metastasis-clonal events suggests that colorectal cancer distant metastases may not follow a uniform course of genomic evolution, which should be considered in the genetic diagnosis and the selection of therapeutic targets for the advanced colorectal cancer. Clin Cancer Res; 21(19); 4461–72. ©2015 AACR.

Although recurrent gene fusions such as JAZF1-JJAZ1 are considered driver events for endometrial stromal sarcoma (ESS) development, other genomic alterations remain largely unknown. In this study, we performed whole-exome sequencing, transcriptome sequencing and copy number profiling for five ESSs (three low-grade ESS (LG-ESS) and two undifferentiated uterine sarcomas (UUSs)). All three LG-ESSs exhibited either one of JAZF1-SUZ12, JAZF1-PHF1 and MEAF6-PHF1 fusions, whereas the two UUSs did not. All ESSs except one LG-ESS exhibited copy number alterations (CNAs), many of which encompassed cancer-related genes. In UUSs, five CNAs encompassing cancer-related genes (EZR, CDH1, RB1, TP53 and PRKAR1A) accompanied their expressional changes, suggesting that they might stimulate UUS development. We found 81 non-silent mutations (35 from LG-ESSs and 46 from UUSs) that included 15 putative cancer genes catalogued in cancer-related databases, including PPARG and IRF4 mutations. However, they were non-recurrent and did not include any well-known mutations, indicating that point mutations may not be a major driver for ESS development. Our data show that gene fusions and CNAs are the principal drivers for LG-ESS and USS, respectively, but both may require additional genomic alterations including point mutations. These differences may explain the different biologic behaviors between LG-ESS and UUS. Our findings suggest that ESS development requires point mutations and CNAs as well as the gene fusions.

Although the colorectal adenoma-to-carcinoma sequence represents a classical cancer progression model, the evolution of the mutational landscape underlying this model is not fully understood. In this study, we analyzed eight synchronous pairs of colorectal high-grade adenomas and carcinomas, four microsatellite-unstable (MSU) and four -stable (MSS) pairs, using whole-exome sequencing. In the MSU adenoma-carcinoma pairs, we observed no subclonal mutations in adenomas that became fixed in paired carcinomas, suggesting a ‘parallel’ evolution of synchronous adenoma-to-carcinoma, rather than a ‘stepwise’ evolution. The abundance of indel (in MSU and MSS pairs) and microsatellite instability (in MSU pairs) was noted in the later adenoma- or carcinoma-specific mutations, indicating that the mutational processes and functional constraints operative in early and late colorectal carcinogenesis are different. All MSU cases exhibited clonal, truncating mutations in ACVR2A, TGFBR2, and DNA mismatch repair genes, but none were present in APC or KRAS. In three MSS pairs, both APC and KRAS mutations were identified as both early and clonal events, often accompanying clonal copy number changes. An MSS case uniquely exhibited clonal ERBB2 amplification, followed by APC and TP53 mutations as carcinoma-specific events. Along with the previously unrecognized clonal origins of synchronous colorectal adenoma-carcinoma pairs, our study revealed that the preferred sequence of mutational events during colorectal carcinogenesis can be context-dependent.

// Seung-Hyun Jung 1, 3, * , Youn Jin Choi 2, * , Min Sung Kim 2 , In-Pyo Baek 1, 3 , Sung Hak Lee 4 , Ah Won Lee 4 , Soo Young Hur 5 , Tae-Min Kim 6 , Sug Hyung Lee 2 , Yeun-Jun Chung 1, 3 1 Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 2 Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 3 Department of Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 4 Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 5 Department of Obstetrics/Gynecology, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea 6 Department of Medical Informatics, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea * These authors have contributed equally to this work Correspondence to: Yeun-Jun Chung, e-mail: yejun@catholic.ac.kr Sug Hyng Lee, e-mail: suhulee@catholic.ac.kr Keywords: Uterine cervix cancer, Cervical intraepithelial neoplasia, Cervical squamous cell carcinoma, Mutation, Copy number alteration Received: November 03, 2014 Accepted: December 20, 2014 Published: January 23, 2015 ABSTRACT Although cervical intraepithelial neoplasia (CIN) is considered a neoplasia, its genomic alterations remain unknown. For this, we performed whole-exome sequencing and copy number profiling of three CINs, a microinvasive carcinoma (MIC) and four cervical squamous cell carcinomas (CSCC). Both total mutation and driver mutation numbers of the CINs were significantly fewer than those of the MIC/CSCCs ( P = 0.036 and P = 0.018, respectively). Importantly, PIK3CA was altered in all MIC/CSCCs by either mutation or amplification, but not in CINs. The CINs harbored significantly lower numbers of copy number alterations (CNAs) than the MIC/CSCCs as well ( P = 0.036). Pathway analysis predicted that the MIC/CSCCs were enriched with cancer-related signalings such as cell adhesion, mTOR signaling pathway and cell migration that were depleted in the CINs. The mutation-based estimation of evolutionary ages identified that CIN genomes were younger than MIC/CSCC genomes. The data indicate that CIN genomes harbor unfixed mutations in addition to human papilloma virus infection but require additional driver hits such as PIK3CA, TP53, STK11 and MAPK1 mutations for CSCC progression. Taken together, our data may explain the long latency from CIN to CSCC progression and provide useful information for molecular diagnosis of CIN and CSCC.

Next-generation sequencing (NGS) is widely used to identify the causative mutations underlying diverse human diseases, including cancers, which can be useful for discovering the diagnostic and therapeutic targets. Currently, a number of single-nucleotide variant (SNV)-calling algorithms are available; however, there is no tool for visualizing the recurrent and phenotype-specific mutations for general researchers. In this study, in order to support defining the recurrent mutations or phenotype-specific mutations from NGS data of a group of cancers with diverse phenotypes, we aimed to develop a user-friendly tool, named mutation arranger for defining phenotype-related SNV (MAP). MAP is a user-friendly program with multiple functions that supports the determination of recurrent or phenotype-specific mutations and provides graphic illustration images to the users. Its operation environment, the Microsoft Windows environment, enables more researchers who cannot operate Linux to define clinically meaningful mutations with NGS data from cancer cohorts.

Next generation sequencing (NGS) has traditionally been performed in various fields including agricultural to clinical and there are so many sequencing platforms available in order to obtain accurate and consistent results. However, these platforms showed amplification bias when facilitating variant calls in personal genomes. Here, we sequenced whole genomes and whole exomes from ten Korean individuals using Illumina and Ion Proton, respectively to find the vulnerability and accuracy of NGS platform in the GC rich/poor area. Overall, a total of 1013 Gb reads from Illumina and ~39.1 Gb reads from Ion Proton were analyzed using BWA-GATK variant calling pipeline. Furthermore, conjunction with the VQSR tool and detailed filtering strategies, we achieved high-quality variants. Finally, each of the ten variants from Illumina only, Ion Proton only, and intersection was selected for Sanger validation. The validation results revealed that Illumina platform showed higher accuracy than Ion Proton. The described filtering methods are advantageous for large population-based whole genome studies designed to identify common and rare variations associated with complex diseases.

Intratumoural heterogeneity (ITH) leads to regional biases of the mutational landscape in a single tumour and may influence the single biopsy-based clinical diagnosis and treatment decision. To evaluate the extent of ITH in unifocal prostate cancers (PCAs), we analysed multiple regional biopsies from three PCAs, using whole-exome sequencing, DNA copy number and gene expression profiling analyses. A substantial level of ITH was identified, in that 0-61% and 18-71% of somatic variants were common or private, respectively, within a given cancer. The enhanced mutation detection rate in the combined sequencing dataset across intratumoural biopsies was demonstrated with respect to the total number of mutations identified in a given tumour. Allele frequencies of the mutations were positively correlated with the levels of intratumoural recurrence (private

Article history: An OrthoMTA compacter is made by machining a Ni-Ti alloy wire using tapered helix creep-feed grinding machines. This aim of this study is to find the optimal dressing conditions to sharpen the corner of a cubic boron nitride (CBN) wheel. On the basis of the results of various experiments, it is verified that the most important factors in dressing are the dressing depth and feeding method, whereas the feed rate has less importance for producing a smaller corner R value. The study also finds the optimum dressing depth to reduce the dressing time, a feeding speed and method to stabilize the machining, and the mesh grade for the CBN wheel to make the groove of the compacter deeper.

The present Common Structural Rules for double hull oil tanker is not included the residual strength, which is one of the functional requirements in design part of Goal-based new ship construction standards (GBS). The GBS will be enforced after July 1, 2016. The requirement related residual strength has the goal to build safe ship even if she has the specified damages due to marine accidents including collision and grounding. In order to assess the residual strength based on risk for structural damages according to GBS, tons of nonlinear FE analysis work taking into account various types of damage will be needed. The Smith`s method, a kind of simplified method for the strength analysis is very useful for this purpose. In this paper, the residual strength assessments based on ultimate strength using Smith`s method were carried out. The objected ship is VLCC with stranding damage in bottom structures. Also, the results were compared with that of nonlinear FE analysis using three cargo hold model.

Early gastric cancers (EGCs) precede advanced gastric cancers (AGCs), with a favourable prognosis compared to AGC. To understand the progression mechanism of EGC to AGC, it is required to disclose EGC and AGC genomes in mutational and evolutionary perspectives. We performed whole-exome sequencing and copy number profiling of nine microsatellite (MS)-unstable (MSI-H) (five EGCs and four AGCs) and eight MS-stable (MSS) gastric cancers (four EGCs and four AGCs). In the cancers, we observed well-known driver mutations (TP53, APC, PIK3CA, ARID1A, and KRAS) that were enriched in cancer-related pathways, including chromatin remodelling and tyrosine kinase activity. The MSI-H genomes harboured ten times more mutations, but were largely depleted of copy number alterations (CNAs) compared to the MSS cancers. Interestingly, EGC genomes showed a comparable level of mutations to AGC in terms of the number, sequence composition, and functional consequences (potential driver mutations and affected pathways) of mutations. Furthermore, the CNAs between EGC and AGC genomes were not significantly different in either MSI-H and MSS. Evolutionary analyses using somatic mutations and MSI as molecular clocks further identified that EGC genomes were as old as AGC genomes in both MSS and MSI-H cancers. Our results suggest that the genetic makeup for gastric cancer may already be achieved in EGC genomes and that the time required for transition to AGC may be relatively short. Also, the data suggest a possibility that the mutational profiles obtained from early biopsies may be useful in the clinical settings for the molecular diagnosis and therapeutics of gastric cancer patients.

Background Although high grade prostatic intraepithelial neoplasia (HGPIN) is considered a neoplastic lesion that precedes prostate cancer (PCA), the genomic structures of HGPIN remain unknown. Objective Identification of the genomic landscape of HGPIN and the genomic differences between HGPIN and PCA that may drive the progression to PCA. Design, settings, and participants We analyzed 20 regions of paired HGPIN and PCA from six patients using whole-exome sequencing and array-comparative genomic hybridization. Outcome measurements and statistical analysis Somatic mutation and copy number alteration (CNA) profiles of paired HGPIN and PCA were measured and compared. Results and limitations The number of total mutations and CNAs of HGPINs were significantly fewer than those of PCAs. Mutations in FOXA1 and CNAs (1q and 8q gains) were detected in both HGPIN and PCA (‘common'), suggesting their roles in early PCA development. Mutations in SPOP , KDM6A , and KMT2D were ‘PCA-specific', suggesting their roles in HGPIN progression to PCA. The 8p loss was either ‘common' or ‘PCA-specific'. In-silico estimation of evolutionary ages predicted that HGPIN genomes were much younger than PCA genomes. Our data show that PCAs are direct descendants of HGPINs in most cases that require more genomic alterations to progress to PCA. The nature of heterogeneous HGPIN population that might attenuate genomic signals should further be studied. Conclusions HGPIN genomes harbor relatively fewer mutations and CNAs than PCA but require additional hits for the progression. Patient summary In this study, we suggest a systemic diagram from high grade prostatic intraepithelial neoplasia (HGPIN) to prostate cancer (PCA). Our results provide a clue to explain the long latency from HGPIN to PCA and provide useful information for the genetic diagnosis of HGPIN and PCA.