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Background Understanding the genomic determinants associated with metastasis in colorectal cancers (CRCs) provides crucial clues for improving patient care. Patients and methods In this study, we performed whole-exome sequencing as well as RNA sequencing analyses on five pairs of primary and liver metastasized samples from CRC patients together with blood/normal control samples for each pair. Results We identified genomic deletions in the region of 8p21-23 (q value Conclusion Taken together, our findings uncovered various genomic alterations potentially leading to metastasis in CRC and provide important insights into the development of potential therapeutic targets for preventing metastatic progression of CRC.

The etiologies and prevalence of sporadic, postlingual-onset, progressive auditory neuropathy spectrum disorder (ANSD) have rarely been documented. Thus, we aimed to evaluate the prevalence and molecular etiologies of these cases. Three out of 106 sporadic progressive hearing losses turned out to manifest ANSD. Through whole exome sequencing and subsequent bioinformatics analysis, two out of the three were found to share a de novo variant, p.E818K of ATP1A3, which had been reported to cause exclusively CAPOS (cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss) syndrome. However, hearing loss induced by CAPOS has never been characterized to date. Interestingly, the first proband did not manifest any features of CAPOS, except subclinical areflexia; however, the phenotypes of second proband was compatible with that of CAPOS, making this the first reported CAPOS allele in Koreans. This ANSD phenotype was compatible with known expression of ATP1A3 mainly in the synapse between afferent nerve and inner hair cells. Based on this, cochlear implantation (CI) was performed in the first proband, leading to remarkable benefits. Collectively, the de novo ATP1A3 variant can cause postlingual-onset auditory synaptopathy, making this gene a significant contributor to sporadic progressive ANSD and a biomarker ensuring favorable short-term CI outcomes.

Stargardt-like macular dystrophy 4 (STGD4) is a rare macular dystrophy characterized by bull's eye atrophy of the macula and the underlying retinal pigment epithelium. Patients with STGD4 show decreased central vision, which often progresses to severe vision loss. The PROM1 gene encodes prominin-1, which is a 5-transmembrane glycoprotein also known as CD133 and is involved in photoreceptor disk morphogenesis. PROM1 mutations have been identified as genetic causes for STGD4 and other retinal degenerations such as retinitis pigmentosa. We report a case of STGD4 with a PROM1 p.R373C mutation in a Korean patient. Ophthalmic examinations of a 38-yr old man complaining of decreased visual acuity revealed bilateral atrophic macular lesions consistent with STGD4. Targeted exome sequencing of known inherited retinal degeneration genes revealed a heterozygous missense mutation c.1117C>T (p.R373C) of PROM1, which was confirmed by Sanger sequencing. To the best of our knowledge, this is the first case of a PROM1 mutation causing STGD4 in Koreans.

Accurate detection of genomic alterations using high-throughput sequencing is an essential component of precision cancer medicine. We characterize the variant allele fractions (VAFs) of somatic single nucleotide variants and indels across 5095 clinical samples profiled using a custom panel, CancerSCAN. Our results demonstrate that a significant fraction of clinically actionable variants have low VAFs, often due to low tumor purity and treatment-induced mutations. The percentages of mutations under 5% VAF across hotspots in EGFR, KRAS, PIK3CA, and BRAF are 16%, 11%, 12%, and 10%, respectively, with 24% for EGFR T790M and 17% for PIK3CA E545. For clinical relevance, we describe two patients for whom targeted therapy achieved remission despite low VAF mutations. We also characterize the read depths necessary to achieve sensitivity and specificity comparable to current laboratory assays. These results show that capturing low VAF mutations at hotspots by sufficient sequencing coverage and carefully tuned algorithms is imperative for a clinical assay., High-throughput sequencing is used to identify somatic variants in cancer patients. Here, the authors perform panel-based profiling of 5095 clinical samples and demonstrate that many clinically-actionable variants have low variant allele fractions, requiring assays with high detection sensitivity.

Purpose Patient-derived tumor xenografts (PDXs) can provide more reliable information about tumor biology than cell line models. We developed PDXs for epithelial ovarian cancer (EOC) that have histopathologic and genetic similarities to the primary patient tissues and evaluated their potential for use as a platform for translational EOC research. Materials and methods We successfully established PDXs by subrenal capsule implantation of primary EOC tissues into female BALB/C-nude mice. The rate of successful PDX engraftment was 48.8% (22/45 cases). Hematoxylin and eosin staining and short tandem repeat analysis showed histopathological and genetic similarity between the PDX and primary patient tissues. Results Patients whose tumors were successfully engrafted in mice had significantly inferior overall survival when compared with those whose tumors failed to engraft (p=0.040). In preclinical tests of this model, we found that paclitaxel-carboplatin combination chemotherapy significantly deceased tumor weight in PDXs compared with the control treatment (p=0.013). Moreover, erlotinib treatment significantly decreased tumor weight in epidermal growth factor receptor-overexpressing PDX with clear cell histology (p=0.023). Conclusion PDXs for EOC with histopathological and genetic stability can be efficiently developed by subrenal capsule implantation and have the potential to provide a promising platform for future translational research and precision medicine for EOC.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK-rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule-associated protein-like 4 (EML4)-ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK-rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4-ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4-ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non-homologous end-joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4-ALK variants, new ALK somatic mutations, and novel ALK-fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Choroideremia is a rare X-linked disorder causing progressive chorioretinal atrophy. Affected patients develop night blindness with progressive peripheral vision loss and eventual blindness. Herein, we report two Korean families with choroideremia. Multimodal imaging studies showed that the probands had progressive loss of visual field with characteristic chorioretinal atrophy, while electroretinography demonstrated nearly extinguished cone and rod responses compatible with choroideremia. Sanger sequencing of all coding exons and flanking intronic regions of the CHM gene revealed a novel small deletion at a splice site (c.184_189+3delTACCAGGTA) in one patient and a deletion of the entire exon 9 in the other. This is the first report on a molecular genetic diagnosis of choroideremia in Korean individuals. Molecular diagnosis of choroideremia should be widely adopted for proper diagnosis and the development of new treatment modalities including gene therapy.

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.

The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT-mutated melanoma.

To generate accurate next-generation sequencing (NGS) data, the amount and quality of DNA extracted is critical. We analyzed 1564 tissue samples from patients with metastatic or recurrent solid tumor submitted for NGS according to their sample size, acquisition method, organ, and fixation to propose appropriate tissue requirements. Of the 1564 tissue samples, 481 (30.8%) consisted of fresh-frozen (FF) tissue, and 1,083 (69.2%) consisted of formalin-fixed paraffin-embedded (FFPE) tissue. We obtained successful NGS results in 95.9% of cases. Out of 481 FF biopsies, 262 tissue samples were from lung, and the mean fragment size was 2.4 mm. Compared to lung, GI tract tumor fragments showed a significantly lower DNA extraction failure rate (2.1 % versus 6.1%, p = 0.04). For FFPE biopsy samples, the size of biopsy tissue was similar regardless of tumor type with a mean of 0.8 × 0.3 cm, and the mean DNA yield per one unstained slide was 114 ng. We obtained highest amount of DNA from the colorectum (2353 ng) and the lowest amount from the hepatobiliary tract (760.3 ng) likely due to a relatively smaller biopsy size, extensive hemorrhage and necrosis, and lower tumor volume. On one unstained slide from FFPE operation specimens, the mean size of the specimen was 2.0 × 1.0 cm, and the mean DNA yield per one unstained slide was 1800 ng. In conclusions, we present our experiences on tissue requirements for appropriate NGS workflow: > 1 mm2 for FF biopsy, > 5 unstained slides for FFPE biopsy, and > 1 unstained slide for FFPE operation specimens for successful test results in 95.9% of cases.

// Soo Hyun Lee 1, 2, * , Jung-Sun Kim 3, * , Siyuan Zheng 4 , Jason T. Huse 2 , Joon Seol Bae 1 , Ji Won Lee 5 , Keon Hee Yoo 5 , Hong Hoe Koo 5 , Sungkyu Kyung 6 , Woong-Yang Park 1, 7 and Ki W. Sung 5 1 Samsung Genome Institute, Samsung Medical Center, Seoul, Republic of Korea 2 Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4 Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 6 Department of Bioinformatics, Sungsil University, Seoul, Republic of Korea 7 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence to: Woong-Yang Park, email: woongyang.park@samsung.com Ki W. Sung, email: kwsped@skku.edu Keywords: ARID1B, ALK, MYCN, neuroblastoma, sequencing Received: July 02, 2016 Accepted: April 11, 2017 Published: April 28, 2017 ABSTRACT Targeted panel sequencing was performed to determine molecular targets and biomarkers in 72 children with neuroblastoma. Frequent genetic alterations were detected in ALK (16.7%), BRCA1 (13.9%), ATM (12.5%), and PTCH1 (11.1%) in an 83-gene panel. Molecular targets for targeted therapy were identified in 16 of 72 patients (22.2%). Two-thirds of ALK mutations were known to increase sensitivity to ALK inhibitors. Sequence alterations in ARID1B were identified in 5 of 72 patients (6.9%). Four of five ARID1B alterations were detected in tumors of high-risk patients. Two of five patients with ARID1B alterations died of disease progression. Relapse-free survival was lower in patients with ARID1B alterations than in those without ( p = 0.01). In analysis confined to high-risk patients, 3-year overall survival was lower in patients with an ARID1B alteration (33.3 ± 27.2%) or MYCN amplification (30.0 ± 23.9%) than in those with neither ARID1B alteration nor MYCN amplification (90.5 ± 6.4%, p = 0.05). These results provide possibilities for targeted therapy and a new biomarker identifying a subgroup of neuroblastoma patients with poor prognosis.

Hidradenocarcinoma is an extremely rare cutaneous skin carcinoma which arise from the sweat gland. There are several report about potential targets in this rare disease including estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). A 53-year-old man who presented with a slowly growing mass on the left thumb which was pathologically confirmed of HER2 negative, ER negative hidradenocarcinoma. At the time of presentation, the patient had metastatic lesions to lung and axillary lymph nodes. The patient received several lines of palliative chemotherapy and radiotherapy, but died of the disease progression. We identified TP53 G245S mutation in both primary tumor specimen and cell-free based targeted sequencing. Herein, we report on a very rare case of metastatic hidradenocarcinoma with TP53 G245S mutation but no other treatable targets from clinical sequencing, which pursued an aggressive clinical course.

Precision medicine in cancer proposes that genomic characterization of tumors can inform personalized targeted therapies. However, this proposition is complicated by spatial and temporal heterogeneity. Here we study genomic and expression profiles across 127 multisector or longitudinal specimens from 52 individuals with glioblastoma (GBM). Using bulk and single-cell data, we find that samples from the same tumor mass share genomic and expression signatures, whereas geographically separated, multifocal tumors and/or long-term recurrent tumors are seeded from different clones. Chemical screening of patient-derived glioma cells (PDCs) shows that therapeutic response is associated with genetic similarity, and multifocal tumors that are enriched with PIK3CA mutations have a heterogeneous drug-response pattern. We show that targeting truncal events is more efficacious than targeting private events in reducing the tumor burden. In summary, this work demonstrates that evolutionary inference from integrated genomic analysis in multisector biopsies can inform targeted therapeutic interventions for patients with GBM.

// Ji-Yeon Kim 1, * , Eunjin Lee 2, * , Kyunghee Park 2 , Woong-Yang Park 2 , Hae Hyun Jung 4 , Jin Seok Ahn 1 , Young-Hyuck Im 1, 3 , Yeon Hee Park 1, 3, 4 1 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea 2 Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea 3 Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea 4 Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea * These authors contributed equally to this work Correspondence to: Yeon Hee Park, email: yhparkhmo@skku.edu Keywords: metastatic, breast cancer, TP53, PIK3CA Received: September 09, 2016 Accepted: February 20, 2017 Published: March 03, 2017 ABSTRACT Breast cancer (BC) has been genetically profiled through large-scale genome analyses. However, the role and clinical implications of genetic alterations in metastatic BC (MBC) have not been evaluated. Therefore, we conducted whole-exome sequencing (WES) and RNA-Seq of 37 MBC samples and targeted deep sequencing of another 29 MBCs. We evaluated somatic mutations from WES and targeted sequencing and assessed gene expression and performed pathway analysis from RNA-Seq. In this analysis, PIK3CA was the most commonly mutated gene in estrogen receptor (ER)-positive BC, while in ER-negative BC, TP53 was the most commonly mutated gene ( p = 0.018 and p < 0.001, respectively). TP53 stopgain/loss and frameshift mutation was related to low expression of TP53 in contrast nonsynonymous mutation was related to high expression. The impact of TP53 mutation on clinical outcome varied with regard to ER status. In ER-positive BCs, wild type TP53 had a better prognosis than mutated TP53 (median overall survival (OS) (wild type vs. mutated): 88.5 ± 54.4 vs. 32.6 ± 10.7 (months), p = 0.002). In contrast, mutated TP53 had a protective effect in ER-negative BCs (median OS: 0.10 vs. 32.6 ± 8.2, p = 0.026). However, PIK3CA mutation did not affect patient survival. In gene expression analysis, CALM1 , a potential regulator of AKT , was highly expressed in PIK3CA -mutated BCs. In conclusion, mutation of TP53 was associated with expression status and affect clinical outcome according to ER status in MBC. Although mutation of PIK3CA was not related to survival in this study, mutation of PIK3CA altered the expression of other genes and pathways including CALM1 and may be a potential predictive marker of PI3K inhibitor effectiveness.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Additional file 1: Figure S1. A schematic illustration on identification of MA-TAM signature and master regulators. Figure S2. MA-TAM target gene pathway enrichment. Figure S3. Global regulatory network of MA-TAM. Figure S4. Multi-color immunohistochemical images of MA-TAM encoding molecules. Figure S5. Effects of MARCO and CCL7 on mesenchymal markers. Figure S6. Effects of TAM-derived CM on GSC stemness in response to irradiation. Figure S7. Effects of anti-MARCO therapeutic antibodies. Figure S8. In vivo effects of MARCOhigh TAMs in PDX models. Figure S9. Clinical correlation of MA-TAM master regulators. Figure S10. Single cell analysis of MA-TAM signature. Figure S11. Transcriptome analysis of scTHI at single-cell resolution. Figure S12. Cytokine array-based characterization of MARCOhigh TAMs. Figure S13. Anatomical expression of MA-TAM signature.

Additional file 2. Frequency of mutated genes in risk groups and list of risk group associated pathway.

Additional file 8. Review history.

Additional file 1. Frequency of variants and mutated genes.

Additional file 5. Result of GSVA analysis.

Additional file 3. Sequence of recurrent fusions and expression profile of genes having fusions.

Additional file 2. Review History.

Additional file 1: Fig. S1. Sample information and upstream intergenic breakpoint distances (UIB). Fig. S2. Additional analysis of IGF2BP3UIB fusions. Fig. S3. Pathway analysis for OR1D4-, CHI3L1- and FUT5-overexpressing breast cancers. Fig. S4. Pathway analysis for LIPG-overexpressing breast cancers and LIPG expression in basal and non-basal breast cancer. Fig. S5. Pathway analysis for LEP-, KY- and FAM107A-overexpressing breast cancers and FAM107A expression in basal and non-basal breast cancer. Fig. S6. Experimental validation. Fig. S7. Cancer genes differentially expressed in IGF2BP3UIB fusion-positive vs -negative groups in thyroid cancer. Fig. S8. Five geneUIB fusions with fixed 5′ partners.

Additional file 4. Result of DEG analysis.

Additional file 6. Result of Mutational signature analysis.

gBRCA1/2 mutations increase the incidence of breast cancer by interrupting the homologous recombination repair (HRR) pathway. Although gBRCA1 and gBRCA2 breast cancer have similar clinical profiles, different molecular characteristics have been observed. In this study, we conducted comprehensive genomic analyses and compared gBRCA1/2 breast cancer. Sanger sequencing to identify gBRCA1/2 mutations was conducted in 2,720 patients, and gBRCA1 (n = 128) and gBRCA2 (n = 126) mutations were analyzed. Within this population, deep target sequencing and matched whole-transcriptome sequencing (WTS) results were available for 46 and 34 patients, respectively. An internal database of patients with breast cancer with wild-type gBRCA was used to compile a target sequencing (n = 195) and WTS (n = 137) reference dataset. Three specific mutation sites, p.Y130X (n = 14) and p.1210Afs (n = 13) in gBRCA1 and p.R294X (n = 22) in gBRCA2, were comparably frequent. IHC subtyping determined that the incidence of triple-negative breast cancer was higher among those with a gBRCA1 mutation (71.9%), and estrogen receptor–positive breast cancer was dominant in those with a gBRCA2 mutation (76.2%). gBRCA1/2 mutations were mutually exclusive with PIK3CA somatic mutations (P < 0.05), and gBRCA1 frequently cooccurred with TP53 somatic mutations (P < 0.05). The median tumor mutation burden was 6.53 per megabase (MB) in gBRCA1 and 6.44 per MB in gBRCA2. The expression of AR, ESR1, and PGR was significantly upregulated with gBRCA2 mutation compared with gBRCA1 mutation. gBRCA1 and gBRCA2 breast cancer have similar clinical characteristics, but they have different molecular subtypes, coaltered somatic mutations, and gene expression patterns. Implications: Even though gBRCA1 and gBRCA2 mutations both alter HRR pathways, our results suggest that they generate different molecular characteristics and different mechanisms of carcinogenesis.

Immunotherapy for metastatic colorectal cancer is effective only for mismatch repair-deficient tumors with high microsatellite instability that demonstrate immune infiltration, suggesting that tumor cells can determine their immune microenvironment. To understand this cross-talk, we analyzed the transcriptome of 91,103 unsorted single cells from 23 Korean and 6 Belgian patients. Cancer cells displayed transcriptional features reminiscent of normal differentiation programs, and genetic alterations that apparently fostered immunosuppressive microenvironments directed by regulatory T cells, myofibroblasts and myeloid cells. Intercellular network reconstruction supported the association between cancer cell signatures and specific stromal or immune cell populations. Our collective view of the cellular landscape and intercellular interactions in colorectal cancer provide mechanistic information for the design of efficient immuno-oncology treatment strategies.

Background Genetic sequencing testing has become widely used to inform treatment decisions for advanced non-small cell lung cancer (NSCLC) patients. We analyzed benefits of genetic sequencing testing in real practice. Methods We retrospectively reviewed 209 NSCLC patients who had no EGFR and ALK alterations on routine molecular tests and underwent next-generation targeted DNA sequencing of 380cancer-related genes between November 2013 and October 2016. The median age was 59 years. 96 patients (46%) were never smokers, and 195 patients (93%) had adenocarcinoma. Results Among 209 total patients, 64 patients (31%) demonstrated actionable genetic alterations. 20 had EGFR mutations (6 L858R, 8 exon 19 deletions, 1 L861Q, 1 G719S, 4 exon 20 duplications), 4 ALK fusions, 9 ROS1 fusions, 6 BRAF V600E mutations, 15 RET fusions, 1 MET high-level amplification, 6 MET exon 14 skipping mutations, and 3 ERBB2 exon 20 insertion mutations. Of the 64 patients harboring actionable alterations, 28 patients received therapy targeted to their own actionable alterations (15 EGFR, 3 ALK, 1 ROS1, 8 RET, 1 BRAF). There were significant differences in the overall survival between individuals with no actionable alterations, those with actionable alterations but no targeted therapy, and those with actionable alterations and targeted therapy (20.1 months vs 17.1 months vs 66.2 months, p Conclusion Targeted DNA sequencing testing could provide improved treatment options for some NSCLC patients and a survival benefit to the NSCLC patients with no EGFR and ALK alterations on routine tests that are treated with targeted therapy.

Extranodal NK/T-cell lymphoma is an aggressive lymphoma that is strongly associated with Epstein-Barr virus infection. Although some extranodal NK/T-cell lymphoma patients have shown responses to immune checkpoint blockade, biomarkers for predicting extranodal NK/T-cell lymphoma patient response to immunotherapy have not yet been defined. To understand the tumor immune microenvironment, we analyzed the expression of 579 immune-related genes and characterized the immune cells using immunohistochemistries and in situ hybridization for EBER. Based on comprehensive analyses, we developed an immune subtyping model that classifies extranodal NK/T-cell lymphoma patients into four tumor immune microenvironment subgroups using three immunohistochemical markers (FoxP3, PD-L1, and CD68). The four tumor immune microenvironment subgroups were named immune tolerance, immune evasion-A, immune evasion-B, and immune silenced. The immune tolerance group was characterized by high-Treg counts and was frequently observed in early stage, and nasal extranodal NK/T-cell lymphoma. The immune evasion group showed high cytotoxic T-cell counts and high PD-L1 expression but low Treg counts. In the immune-silenced group, almost all immune responses were exhausted, most patients were at an advanced stage, and had the poorest disease prognosis among the tumor immune microenvironment subgroups. In some patients (n = 3), a shift in the tumor immune microenvironment subgroup classification was observed in sequential biopsies. The response rate to pembrolizumab, an anti-PD-1 antibody, was 100% (1/1) in the immune tolerance group, 60% (3/5) in the immune evasion group, and 0% (0/5) in the immune-silenced group. We classified extranodal NK/T-cell lymphoma into four tumor immune microenvironment subgroups using a new classification system. In conclusion, we propose that the tumor immune microenvironment of extranodal NK/T-cell lymphoma may change during disease progression and may serve as a useful biomarker for immunotherapy.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.

Accurate detection of genomic fusions by high-throughput sequencing in clinical samples with inadequate tumor purity and formalin-fixed, paraffin-embedded tissue is an essential task in precise oncology. We developed the fusion detection algorithm Junction Location Identifier (JuLI) for optimization of high-depth clinical sequencing. Novel filtering steps were implemented to minimize false positives in the clinical setting. The algorithm was comprehensively validated using high-depth sequencing data from cancer cell lines and clinical samples and genome sequencing data from NA12878. JuLI showed improved performance mainly in positive predictive value over state-of-the-art fusion callers in cases with high-depth clinical sequencing and rescued a driver fusion from false negative in plasma cell-free DNA using joint calling.

Diaphanous-related formin 1 (DIA1), which assembles the unbranched actin microfilament and microtubule cytoskeleton, is encoded byHere, we report the first constitutively active mutant in the DID (p.A265S) of humans with only hearing loss and not blood cell abnormality through whole exome sequencing. The previously reported DAD mutants and our DID mutant (p.A265S) shared the finding of diminished autoinhibitory interaction, abnormally upregulated actin polymerisation activity and increased localisations at the plasma membrane. However, the obvious defect in the DIA1-driven assembly of cytoskeleton 'during cell division' was only from the DAD mutants, not from p.A265S, which did not show any blood cell abnormality. We also evaluated the five DID mutants in the hydrophobic pocket since four of these five additional mutants were predicted to critically disrupt interaction between the DID and DAD. These additional pathogenic DID mutants revealed varying degrees of defect in the DIA1-driven cytoskeleton assembly, including nearly normal phenotype during cell division as well as obvious impaired autoinhibition, again coinciding with our key observation in DIA1 mutant (p.A265S) in the DID.Here, we report the first mutant in the DID of humans with only hearing loss. The differential cell biological phenotypes of DIA1 during cell division appear to be potential determinants of the clinical severity of

Neoadjuvant chemotherapy studies have consistently reported a strong correlation between pathologic response and long-term outcome in triple-negative breast cancer (TNBC). We aimed to define minimal gene signatures for predicting chemoresponse by a three-step approach and to further develop a risk-stratification method of TNBC. The first step involved the detection of genes associated with resistance to docetaxel in eight TNBC cell lines, leading to identification of thousands of candidate genes. Through subsequent second and third step analyses with gene set enrichment analysis and survival analysis using public expression profiles, the candidate gene list was reduced to prognostic core gene signatures comprising ten or four genes. The prognostic core gene signatures include three up-regulated (CEBPD, MMP20, and WLS) and seven down-regulated genes (ASF1A, ASPSCR1, CHAF1B, DNMT1, GINS2, GOLGA2P5, and SKA1). We further develop a simple risk-stratification method based on expression profiles of the core genes. Relative expression values of the up-regulated and down-regulated core genes were averaged into two scores, Up and Down scores, respectively; then samples were stratified by a diagonal line in a xy plot of the Up and Down scores. Based on this method, the patients were successfully divided into subgroups with distinct chemoresponse and prognosis. The prognostic power of the method was validated in three independent public datasets containing 230, 141, and 117 TNBC patients with chemotherapy. In multivariable Cox regression analysis, the core gene signatures were significantly associated with prognosis independent of tumor stage and age at diagnosis. In meta-analysis, we found that five core genes (CEBPD, WLS, CHAF1B, GINS2, and SKA1) play opposing roles, either tumor promoter or suppressor, in TNBC and non-TNBC tumors respectively, depending on estrogen receptor status. The results may provide a promising prognostic tool for predicting chemotherapy responders among TNBC patients prior to initiation of chemotherapeutic treatment.

Tumor genetic heterogeneity may underlie poor clinical outcomes because diverse subclones could be comprised of metastatic and drug resistant cells. Targeted deep sequencing has been used widely as a diagnostic tool to identify actionable mutations in cancer patients. In this study, we evaluated the clinical utility of estimating tumor heterogeneity using targeted panel sequencing data. We investigated the prognostic impact of a tumor heterogeneity (TH) index on clinical outcomes, using mutational profiles from targeted deep sequencing data acquired from 1,352 patients across 8 cancer types. The TH index tended to be increased in high pathological stage disease in several cancer types, indicating clonal expansion of cancer cells as tumor progression proceeds. In colorectal cancer patients, TH index values also correlated significantly with clinical prognosis. Integration of the TH index with genomic and clinical features could improve the power of risk prediction for clinical outcomes. In conclusion, deep sequencing to determine the TH index could serve as a promising prognostic indicator in cancer patients.

Objectives Adenocarcinoma (ADC) to squamous cell carcinoma (SCC) transformation (AST) is reported in epidermal growth factor receptor (EGFR)-mutated non–small cell lung cancer after tyrosine kinase inhibitor (TKI) failure. However, little is known about the underlying genomic changes during the AST process. Materials and methods We retrospectively reviewed our tissue database collected after first- or second- generation EGFR TKI resistance (n = 263) and identified 3 cases of AST. The additional case was acquired from the osimertinib resistance sample. Deep target sequencing (381 genes) using paired samples from 4 patients with AST after EGFR TKI treatment was performed. The histology of each sample was confirmed by TTF-1 and p63 immunohistochemistry. The patients received first- or second-generation EGFR TKI as an initial treatment. Results Overall incidence of AST was 1.1% (3/263). Transformed SCC acquired genomic alterations related to the PI3K/AKT/mTOR pathway, in addition to the initial EGFR mutation. In a representative case, two separate sub-clones, with a PTEN nonsense mutation and EGFR p.T790 M mutation, were observed without histologic transformation at the time of gefitinib resistance. After subsequent treatment with osimertinib, SCC transformation was observed with the disappearance of the EGFR p.T790 M mutation and acquired copy number loss in PTEN. Adopting the sub-clonal fraction model elucidates the sub-clonal evolution process of the PTEN mutant sub-clone toward AST under the background of EGFR mutation. The rest of the transformed samples also had acquired genomic alterations in PTEN, LKB1, PIK3CA, or RICTOR, which are related to the PI3K/AKT/mTOR pathway. Conclusions Paired genomic analysis from our sample provides early clinical evidence of the ADC to SCC lineage transition that might be provoked by an alteration in the PI3K/AKT/mTOR pathway during EGFR TKI treatment. This finding could potentially broaden the known spectrum of EGFR TKI resistance mechanisms.

557 Background: To precisely predict neoadjuvant chemotherapy (NAC) response and long-term prognosis, we developed prediction model with clinical and genomic characteristics of breast cancer (BC). Methods: We included early and locally advanced BC that would be scheduled to receive standard NAC (four cycles of anthracycline and cyclophosphamide and four cycles of docetaxel or docetaxel plus trastuzumab for HER2+ BC) followed by curative surgery. For each patient, tumor tissue and matched blood were prospectively collected three times: at diagnosis (T1), three weeks after the first cycle of chemotherapy (T2), and curative surgery (T3). Whole exome sequencing (WES) was performed to detect somatic mutation, mutational signature and tumor mutational burden (TMB) while RNASeq with PAM50 prediction was to classify intrinsic subtype. In terms of clinical variables, clinical stage and IHC subtype at diagnosis, residual cancer burden (RCB) class and distant recurrence free survival (DRFS) were used. Logistic regression was used for predicting RCB class with clinical and genomic variables at T1. Univariate and multivariate Cox regression were performed to identify prognostic factors for DRFS. Results: In total, 210 patients were enrolled and treated with NAC as scheduled. We successfully conducted WES in 231 BC tissues (T1:117, T2:101 and T3:13) from 117 patients. In NAC response, 13 patients were in RCB class 3, 39 in class 2, 14 in class 1 and 46 in class 0. Median follow up duration was 44months and distant recurrence was observed in 13 patients. TP53 mutation (68%) was the most commonly detected genetic alteration. ARID1A, CDH1, CSMD3, LRP1B, PIK3CA, RUNX1 and TP53 were significantly mutated genes in driver gene analysis. Median TMB was 87 (range, 14-570) and signature 3 was most frequently observed. Among genetic characteristics, high TMB was significantly associated with better NAC response compared with low TMB (hazard ratio[HR] for RCB class III: 0.11, 95% confident interval[CI]: 0.01, 0.74, p = 0.05). In prediction model, combination of seven variables: intrinsic subtype, TMB, LRRK1, OPLAH, and PIK3CA hotspot mutation, ERBB2 amplification, and clinical stage had 0.83 in area under curve (AUC) and 0.75 in accuracy. High clinical stage, PTEN and PIK3CA hotspot mutation negatively affected to DRFS while high TMB had protective effect (all ps < 0.05). Prediction model made with five variables: intrinsic subtype, TMB, PTEN mutation, PIK3CA hotspot mutation and clinical stage had 0.88 in c-index (95% CI: 0.81, 0.95). Conclusions: TMB, PIK3CA hotspot mutation and clinical stage showed predictive roles on NAC response and distant recurrence of BC in NAC setting. In prediction model, intrinsic subtype, TMB, LRRK1, OPLAH, and PIK3CA hotspot mutation, ERBB2 amplification, and clinical stage affected to RCB class while intrinsic subtype, TMB, PTEN, PIK3CA hotspot mutation and clinical stage did to DRFS. Clinical trial information: NCT02591966.

1013 Background: The development of CDK4/6 inhibitors represents a significant advance in the treatment of metastatic breast cancer (MBC). To better understand the impact of treatment and drug resistance at the molecular level, we performed multi-omics profiling of matched pre-treatment, on-treatment, and post-progression tumor biopsies from patients treated with palbociclib combined with endocrine blockades (AIs and fulvestrant). The purpose of the study was to identify biomarkers of palbociclib resistance as well as to assess molecular changes during treatment, and those appearing at disease progression. Methods: Patients with Hormone Receptor positive (HR+)/HER2- MBC treated with palbociclib in combination with endocrine therapies were prospectively enrolled from July 2017 to June 2020. Of the 89 patients enrolled, we obtained tumor biopsies and matched blood samples, taken at pre-treatment, on-treatment (6 weeks or 12 weeks) and progressive disease (PD) from 71 patients (first line: 55, second line or later: 16 pts.) who had agreed to informed consent form. Tumor biopsies were profiled using whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-Seq). Results: Median follow-up duration was 20 (3-48) months and median age of the patients was 45 (range 30-71) years. The median progression free survival (PFS) of 71 patients was 15 (0.7-39.8) months. The Luminal B subtype is associated with shorter PFS compared to Luminal A (8.6 m vs 19.3 m, p=0.03, HR=1.96). The Luminal B subtype along with multiple cell cycle regulatory genes such as CCNE1 (8.3 m, p=0.015, HR=2.07), CCNE2 (8.5 m, p=8.5e-3, HR=2.2), CDK2 (8.45 m, p=0.05, HR=1.79) are associated with shorter PFS while estrogen response signatures (20.9 m, p=4.6e-3, HR=0.43) and PGR gene expression (20.2 m, p=6.2e-2, HR=0.56) are associated with more favorable prognosis. A Cox-regression multivariate model (p=5.17e-5, C-Index=0.72) was developed and revealed that PFS is independently associated with BRCA1/2 (HR=1.44; CI=[0.49, 4.29]), TP53 mutation statuses (HR=3.58; CI=[1.58, 8.13]), the HRD mutation signature (HR=1.40; CI=[1.09, 1.81]) and the proliferative index (HR=1.53; CI=[1.00, 2.34]), an expression signature of cell proliferation. Tumors classified as Luminal A at baseline frequently switched into Luminal B or Her2-enriched subtypes at PD, along with up-regulation of cell cycle markers and proliferation signatures. Further, tumor mutation burden (TMB) and HRD index, a DNA-based measure of genomic instability, significantly increased from baseline to PD in patients with TP53 and BRCA1/2 wild-type tumors. Conclusions: Our longitudinal multi-omics study identified prognostic biomarkers as well as post-treatment enrichment of HRD related genomic scars and frequent switching into molecular subtypes with aggressive and estrogen independence characteristics. Clinical trial information: NCT03401359 .

8562 Background: The molecular profiling of small cell lung cancer (SCLC) has demonstrated a high incidence of genomic alterations in cell cycle-related genes and DNA damage and response (DDR) pathways, which correlates with devastating clinical outcomes of SCLC. Using two small molecules targeting the DNA repair pathway, olaparib (PARP inhibitor) and ceralasertib (ATR inhibitor), we evaluated their clinical efficacy in monotherapy or in combination, in relapsed SCLC. Methods: As part of the phase II biomarker-driven umbrella study in SCLC (SUKSES), patients who failed prior platinum-based regimen were enrolled and allocated based on their genomic alterations. Patients with mutations harboring HR pathway gene mutation not limited to BRCA 1 or 2, ATM deficiency or MRE11A mutations were allocated to the olaparib monotherapy (SUKSES-B, NCT03009682). As an biomarker non-matched arm, biomarker unselected patients were also allowed to receive olaparib and ceralasertib arm (SUKSES-N2, NCT0328607). The primary endpoint was objective response rate (ORR), and two-stage Simon’s design was used. Results: Based on pre-defined protocol criteria, both arms terminated at stage 1. In the olaparib monotherapy arm (SUKSES-B, n = 15), ORR was 6.7%, and disease control rate (DCR) was 26.7%, 1 partial response (PR), and 3 stable diseases (SD). Median progression-free survival (PFS) was 1.25 months (95% confidential interval [CI] 1.18–NA), and median overall survival (OS) was 8.56 months (95% CI, 6.62–NA). Adverse events that led to treatment discontinuation (n = 2 total, 13.3%) were drug related grade 3 renal impairment, thrombocytopenia, and grade 2 anemia. A patient with confirmed PR showed a tumor volume decrease of 37% compared to the baseline, and a splicing site mutation in BRCA2 was identified from deep target sequencing. In the olaparib and ceralasertib treatment arm (SUKSES-N2, n = 26), ORR was 3.8% (n = 1) and DCR was 42.3% (n = 11). Median PFS was 2.75 months (95% CI 1.77–5.44), and OS was 7.18 months (95% CI 5.97-10.79). Three patients discontinued treatment due to drug related grade 5 pneumonia, grade 3 drug-induced pneumonitis and grade 2 anemia. The most common adverse events for the combination were anemia (n = 11, 42.3%), followed by thrombocytopenia (n = 6, 23.1%). A patient with confirmed PR with a 43% decreases in tumor volume compared with baseline had a mutation in TP53. Conclusions: Targeting the DDR pathways with olaparib as single agent or in combination with ceralasertib demonstrated early signal of efficacy in relapsed SCLC patients with a tolerable safety profile. Clinical trial information: NCT03009682, NCT0328607.

Ocular marginal zone lymphoma is a common type of low-grade B-cell lymphoma. To investigate the genomic changes that occur in ocular marginal zone lymphoma, we analyzed 10 cases of ocular marginal zone lymphoma using whole-genome and RNA sequencing and an additional 38 cases using targeted sequencing. Major genetic alterations affecting genes involved in nuclear factor (NF)-κB pathway activation (60%), chromatin modification and transcriptional regulation (44%), and B-cell differentiation (23%) were identified. In whole-genome sequencing, the 6q23.3 region containing TNFAIP3 was deleted in 5 samples (50%). In addition, 5 structural variation breakpoints in the first intron of IL20RA located in the 6q23.3 region was found in 3 samples (30%). In targeted sequencing, a disruptive mutation of TNFAIP3 was the most common alteration (54%), followed by mutations of TBL1XR1 (18%), cAMP response element binding proteins (CREBBP) (17%) and KMT2D (6%). All TBL1XR1 mutations were located within the WD40 domain, and TBL1XR1 mutants transfected into 293T cells increased TBL1XR1 binding with nuclear receptor corepressor (NCoR), leading to increased degradation of NCoR and the activation of NF-κB and JUN target genes. This study confirms genes involving in the activation of the NF-kB signaling pathway is the major driver in the oncogenesis of ocular MZL.

// Sun Young Kim 1, * , Taejin Ahn 2, * Heejin Bang 3, * Jun Soo Ham 1 , Jusun Kim 1 , Seung Tae Kim 1 , Jiryeon Jang 1 , Moonhee Shim 1 , So Young Kang 3 , Se Hoon Park 1 , Byung Hoon Min 4 , Hyuk Lee 4 , Won Ki Kang 1 , Kyoung-Mee Kim 3 , Woongyang Park 2, 5 , Jeeyun Lee 1 1 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Samsung Genome Institute, Seoul, Korea 3 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 4 Division of Gastroenterology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Seoul, Korea * These authors are contributed equally to this work Correspondence to: Jeeyun Lee, email: jyunlee@skku.edu Keywords: gastric cancer, FGFR2 amplification, targeted therapy Received: October 17, 2016 Accepted: January 11, 2017 Published: January 21, 2017 ABSTRACT Background: Fibroblast growth factor 2 ( FGFR2 ) amplification, occurring in ~2–9% of gastric cancers (GC), is associated with poor overall survival. Results: RNA sequencing identified a novel FGFR2-ACSL5 fusion in the resistant tumor that was absent from the matched pre-treatment tumor. The FGFR2 -amplified PDC line was sensitive to FGFR inhibitors whereas the PDC line with concomitant FGFR2 amplification and FGFR2-ACSL5 fusion exhibited resistance. Additionally, the FGFR2 -amplified GC PDC line, which was initially sensitive to FGFR2 inhibitors, subsequently also developed resistance. Materials and Methods: We identified an FGFR2 -amplified patient with GC, who demonstrated a dramatic and long-term response to LY2874455, a pan-FGFR inhibitor, but eventually developed an acquired LY2874455 resistance. Following resistance development, an endoscopic biopsy was performed for transcriptome sequencing and patient-derived tumor cell line (PDC) establishment to elucidate the underlying molecular alterations. Conclusions: FGFR inhibitors may function against FGFR2 -amplified GC, and a novel FGFR2-ACSL5 fusion identified by transcriptomic characterization may underlie clinically acquired resistance. Implications for Practice: Poor treatment response represents a substantial concern in patients with gastric cancer carrying multiple FGFR2 gene copies. Here, we show the utility of a general FGFR inhibitor for initial response prior to treatment resistance and report the first characterization of a potential resistance mechanism involving an FGFR2-ACSL5 fusion protein.

Subcutaneous and visceral adipose tissues show a different risk effect on metabolic disorders because they have distinct cellular properties. We isolated stem cells from the separate human adipose tissues to investigate that subcutaneous and visceral fat depots have metabolic differences. Adipose-derived stem cells (ASCs) were characterized by immunophenotype and differentiation potentials into adipogenic, osteogenic, and chondrogenic lineages. Although subcutaneous and visceral ASCs (S-ASC and V-ASC) express same surface markers (CD31- , CD34- , CD45- , CD73+ , CD90+ , and CD105+ ) and have differentiation potentials, S-ASCs had higher capacity to proliferate and to differentiate into adipogenic lineage than V-ASCs. Next, we identified that S-ASC and V-ASC were genetically distinct based on microarray analysis. Among a total of 810 genes detected in ASCs of both depots, the differentially expressed genes were involved in energy and lipid metabolism. These data show the existence of the intrinsic difference between S-ASC and V-ASC and suggest the differences of anatomically separated adipose tissue. On the basis of the differentially expressed gene profiles between S-ASC and V-ASC, we suggested significant evidence that adipose tissues originating from different anatomic regions are distinguished at the level of the undifferentiated stem cells such as mature adipocytes. V-ASCs had the upregulated clusters of genes related to lipid biosynthesis and metabolism. By contrast, S-ASCs highly expressed genes involved in DNA-dependent transcription, contributing to proliferation. We provide further insights for ASCs with the different origins to understand fat accumulation and distribution and a possibility of ASCs as a therapeutic target against metabolic disorders or cancer.

Background The symptoms of phosphoribosyl pyrophosphate synthetase 1 (PRPS1) deficiency diseases have been reported to be alleviated by medication. In the present study, we report biochemical data that favor PRPS1 deficiency-related hearing loss as a potential target for pharmaceutical treatment. Methods We recruited 42 probands from subjects aged less than 15 years with a moderate degree of nonsyndromic autosomal-recessive or sporadic sensorineural hearing loss (SNHL) in at least one side. Molecular genetic testing, including targeted exome sequencing (TES) of 129 genes for deafness, and in silico prediction were performed. Results A strong candidate variant (p.A82P) of PRPS1 is co-segregated with SNHL in X-linked recessive inheritance from one Korean multiplex SNHL family. Subsequent measurement of in vitro enzymatic activities of PRPS1 from erythrocytes of affected and unaffected family members, as well as unrelated normal controls, confirmed a pathogenic role of this variant. In detail, compared to normal hearing controls (0.23-0.26 nmol/ml/h), the proband, the affected sibling and their normal hearing mother demonstrated a significantly decreased PRPS1 enzymatic activity (0.07, 0.03 and 0.11 nmol/ml/h, respectively). This novel loss-of-function mutation of PRPS1 (p.A82P) is the ninth and sixth most reported mutation in the world and in Asia, respectively. Conclusions DFNX1 was found to account for approximately 2.4% (1/42) of moderate SNHL in a Korean pediatric population. Confirmation of PRPS1 activity deficiency and an audiologic phenotype that initially begins in a milder form of SNHL, as in our family, should indicate the need for rigorous genetic screening as early as possible.

BRCA2 is a multifunctional tumor suppressor involved in homologous recombination (HR), mitotic checkpoint regulation, and telomere homeostasis. Absence of Brca2 in mice results in progressive shortening of telomeres and senescence, yet cells are prone to neoplastic transformation with elongated telomeres, suggesting that BRCA2 has positive and negative effects on telomere length regulation along the path to tumorigenesis. Using Caenorhabditis elegans as a model, we show here that depletion of BRC-2, an ortholog of BRCA2, paradoxically delays senescence in telomerase-deficient mutant worms. Telomerase-deficient worms (trt-1) exhibit early replication senescence due to short telomeres. It should be noted that worms mutated in brc-2 are not viable as well due to massive genotoxic insults. However, when BRC-2 is depleted by RNA interference in trt-1 mutant worms, the number of generations is unexpectedly increased with telomere length maintained, compared to telomerase mutants. Interestingly, depletion of other HR genes such as rad-51 and rad-54 exhibited similar effects. In worms doubly deficient of telomerase and brc-2, rad-51, or rad-54, extra telomeric C-circles were generated, suggesting that abrogation of HR induces an alteration in telomere environment favorable to illegitimate telomere maintenance when telomerase is absent. Collectively, absence of BRC-2 in telomerase-deficient background first leads to telomere shortening, followed by an induction of an as-yet-unknown telomere maintenance pathway, resulting in delay of senescence. The results have implications in the understanding of dysfunctional BRCA2-associated tumorigenesis.

// Je-Gun Joung 1, * , Sang Yun Ha 5, * , Joon Seol Bae 1 , Jae-Yong Nam 1, 2 , Geum-Youn Gwak 6 , Hae-Ock Lee 1, 4 , Dae-Soon Son 1, 3 , Cheol-Keun Park 5 , Woong-Yang Park 1, 2, 4 1 Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 4 Departments of Molecular Cell Biology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Departments of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 6 Departments of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Cheol-Keun Park, email: ckpark@skku.edu Woong-Yang Park, email: woongyang.park@samsung.com Keywords: hepatocellular carcinoma, dysplastic nodules, whole-exome sequencing, single nucleotide variation, copy number variation Received: October 24, 2015 Accepted: May 17, 2016 Published: July 09, 2016 ABSTRACT Dysplastic nodules are premalignant neoplastic nodules found in explanted livers with cirrhosis. Genetic signatures of premalignant dysplastic nodules (DNs) with concurrent hepatocellular carcinoma (HCC) may provide an insight in the molecular evolution of hepatocellular carcinogenesis. We analyzed four patients with multifocal nodular lesions and cirrhotic background by whole-exome sequencing (WES). The genomic profiles of somatic single nucleotide variations (SNV) and copy number variations (CNV) in DNs were compared to those of HCCs. The number and variant allele frequency of somatic SNVs of DNs and HCCs in each patient was identical along the progression of pathological grade. The somatic SNVs in DNs showed little conservation in HCC. Additionally, CNVs showed no conservation. Phylogenetic analysis based on SNVs and copy number profiles indicated a nonlinear segregation pattern, implying independent development of DNs and HCC in each patient. Thus, somatic mutations in DNs may be developed separately from other malignant nodules in the same liver, suggesting a nonlinear model for hepatocarcinogenesis from DNs to HCC.

Background: Biliary tract cancers (BTCs) are rare and heterogeneous group of tumors classified anatomically into intrahepatic and extrahepatic bile ducts and gallbladder adenocarcinomas. Patient-derived tumor cell (PDC) models with genome analysis can be a valuable platform to develop a method to overcome the clinical barrier on BTCs. Material and Methods: Between January 2012 and June 2015, 40 BTC patients’ samples were collected. PDCs were isolated and cultured from surgical specimens, biopsy tissues, or malignant effusions including ascites and pleural fluid. Genome analysis using targeted panel sequencing as well as digital multiplexed gene analysis was applied to PDCs as well as primary tumors. Results: Extrahepatic cholangiocarcinoma (N = 15, 37.5%), intrahepatic cholangiocarcinoma (N = 10, 25.0%), gallbladder cancer (N = 14, 35.0%), and ampulla of Vater cancer (N = 1, 2.5%) were included. We identified 15 mutations with diverse genetic alterations in 19 cases of BTC from primary tumor specimens. The most common molecular alterations were in TP53 (8/19, 42.1%), including missense mutations such as C242Y, E285K, G112S, P19T, R148T, R248Q, and R273L. We also detected two NRAS mutations (G12C and Q61L), two KRAS mutations (G12A and G12S), two ERBB2 mutations (V777L and pM774delinsMA) and amplification, and three PIK3CA mutations (N345K, E545K, and E521K). PDC models were successfully established in 27 of 40 samples (67.5%), including 22/24 from body fluids (91.7%) and 5/16 from tissue specimens (31.3%). Conclusions: PDC models are promising tools for uncovering driver mutations and identifying rational therapeutic strategies in BTC. Application of this model is expected to inform clinical trials of drugs for molecular-based targeted therapy.

The purpose of this study was to evaluate the clinical utility of targeted exome sequencing (TES) as a molecular diagnostic tool for patients with skeletal dysplasia. A total of 185 patients either diagnosed with or suspected to have skeletal dysplasia were recruited over a period of 3 years. TES was performed for 255 genes associated with the pathogenesis of skeletal dysplasia, and candidate variants were selected using a bioinformatics analysis. All candidate variants were confirmed by Sanger sequencing, correlation with the phenotype, and a cosegregation study in the family. TES detected “confirmed” or “highly likely” pathogenic sequence variants in 74% (71 of 96) of cases in the assured clinical diagnosis category and 20.3% (13 of 64 cases) of cases in the uncertain clinical diagnosis category. TES successfully detected pathogenic variants in all 25 cases of previously known genotypes. The data also suggested a copy-number variation that led to a molecular diagnosis. This study demonstrates the feasibility of TES for the molecular diagnosis of skeletal dysplasia. However, further confirmation is needed for a final molecular diagnosis, including Sanger sequencing of candidate variants with suspected, poorly captured exons. Genet Med 18 6, 563–569.

Distant metastases uncommonly occur in differentiated thyroid carcinoma (DTC), but they are a frequent cause of thyroid cancer-related death. Genomic alterations in metastatic tumors, and the relationship with their corresponding primary tumors in DTC, are poorly understood. The objective of this study was to investigate whether genetic alterations in primary tumors are concordant with distant metastases in DTC patients.Surgical samples from primary and matched distant metastatic tumor pairs from 17 DTC patients, and three additional unpaired metastatic tumors from two patients, were analyzed using targeted next-generation sequencing (Ion Torrent Ampliseq cancer panel) with a focus on known recurrent somatic mutations in thyroid cancer. Additionally, TERT promoter mutations were assessed by direct sequencing.BRAF mutations were found in 8/10 patients with papillary thyroid carcinoma (PTC). A NRAS mutation was detected in one patient with follicular variant PTC. TERT promoter mutations were detected in 8/10 patients with PTC, and most were coexistent with a BRAF mutation (7/8 BRAF-positive PTC patients, and one BRAF-negative PTC patient). In follicular thyroid carcinoma, NRAS was the most frequently observed mutation (4/9 patients), followed by HRAS (two patients) and KRAS (one patient). TERT promoter mutations were found in 6/7 RAS-positive follicular thyroid carcinoma patients. Key somatic alterations such as BRAF and RAS mutations were highly concordant between primary and matched metastatic tumors without discrepancies. The BRAF or RAS mutant allelic frequency was higher in matched metastatic tumors than in the corresponding primary tumors (35% vs. 25% for BRAF mutation, p = 0.04; and 40% vs. 34% for RAS mutation, p = 0.002). TERT promoter mutations were also mostly concordant in matched tumors (concordance rate 93%).BRAF, RAS, and TERT mutations are highly prevalent in metastatic DTC, and are concordant between primary and metastatic DTC. This high concordance suggests that primary tumors may reflect the key somatic alterations in matched metastatic DTC. Frequent coexistent TERT promoter and BRAF or RAS mutations in metastatic DTC also suggests its important role in the progression of DTC.