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Background: Studies investigating the relationship between germline telomere length and the clinical characteristics of tumors are very limited. This study evaluated the relationship between germline telomere length and the clinical characteristics of neuroblastoma. In addition, a genome-wide association study (GWAS) was performed to investigate the genetic factors associated with telomere length. Methods: The telomere length of peripheral blood mononuclear cells from 186 patients with neuroblastoma was measured by quantitative PCR. The association between germline telomere length and clinical characteristics, including long-term survival, was investigated. For the GWAS, genotyping was performed with a high-density Illumina bead chip. After strict quality control checks of the samples, an association analysis was conducted. Results: An extra-abdominal primary site, lower stage (stage 1/2), MYCN non-amplified tumor, and being in the low-risk group were associated with longer telomere lengths (P = 0.015, 0.007, 0.046 and 0.042, respectively). Patients with longer third telomeres had longer event-free survival than other patients (P = 0.032). In the GWAS, rs2492082, located in the 3’UTR of the HIVEP3 gene, showed the highest association with telomere length (P = 1.7E-06). Patients with long telomeres had a recessive allele A. A gene-based analysis using the VEGAS2 algorithm showed that the CNTN4 gene had the most significant association with telomere length (P = 1.0E-06). Patients with the CC homozygote in the rs71311728 marker within the CNTN4 gene had a longer telomere length than those with the TC heterozygote. A gene ontology analysis showed that genetically susceptible genes associated with telomere length were mainly distributed in neurite morphogenesis and neuron development.Conclusions: Longer germline telomere length is associated with favorable prognostic factors at diagnosis and eventually better event-free survival in patients with neuroblastoma. In addition, the GWAS demonstrated that genetic markers and genes related to telomere length are associated with neurite morphogenesis and neuron development. A further study with a larger cohort of patients and functional studies are needed.

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.

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions., Understanding the mechanisms that lead to lung adenocarcinoma metastasis is important for identifying new therapeutics. Here, the authors document the changes in the transcriptome of human lung adenocarcinoma using single-cell sequencing and link cancer cell signatures to immune cell dynamics.

Management of heavily pre-treated platinum-resistant ovarian cancer remains a therapeutic challenge. Outcomes are poor with non-platinum, single-agent chemotherapy (CT); however, molecularly targeted anticancer therapies provide new options.This open-label, investigator-initiated, phase 2 umbrella trial (NCT03699449) enrolled patients with platinum-resistant ovarian cancer (at least 2 prior lines of CT and Eastern Cooperative Oncology Group 0/1) to receive combination therapy based on homologous recombination deficiency (HRD) and programmed death ligand 1 (PD-L1) status determined by archival tumour sample assessment. HRD-positive patients were randomised to either olaparib 200mg bid tablet + cediranib 30mg qd (arm 1) or olaparib 300mg bid tablet + durvalumab 1,500mg q4w (arm 2). HRD-negative patients were allocated to either durvalumab 1,500 mg q4w + pegylated liposomal doxorubicin (PLD) or topotecan or weekly paclitaxel (6 cycles; arm 3, those with PD-L1 expression) or durvalumab 1,500 mg q4w + tremelimumab 75mg q4w (4 doses) + PLD or topotecan or weekly paclitaxel (4 cycles; arm 4, those without PD-L1 expression). Arm 5 (durvalumab 1,500 mg q4w + tremelimumab 300mg [1 dose] + weekly paclitaxel [60 mg/m² D1,8,15 q4w for 4 cycles] was initiated after arm 4 completed. The primary endpoint was objective response rate (ORR; Response Evaluation Criteria in Solid Tumours 1.1).Between Dec 2018 and Oct 2020, 70 patients (median 57 years; median 3 prior treatment lines [range 2-10]) were treated (n=16, 14, 5, 18, and 17, respectively). Overall ORR was 37.1% (26/70, 95% confidence interval=25.9, 49.5); 2 achieved complete response. ORR was 50%, 42.9%, 20%, 33.3%, and 29.4%, respectively. Grade 3/4 treatment-related adverse events (TRAEs) were reported in 37.5%, 35.7%, 20%, 66.7%, and 35.3% of patients, respectively. No TRAEs leading to treatment discontinuation and no grade 5 TRAEs were observed.This study, the first biomarker-driven umbrella trial in platinum-resistant recurrent ovarian cancer, suggests clinical utility with biomarker-driven targeted therapy. All treatment combinations were manageable, and without unexpected toxicities.ClinicalTrials.gov Identifier: NCT03699449.

Variant callers typically produce massive numbers of false positives for structural variations, such as cancer-relevant copy-number alterations and fusion genes resulting from genome rearrangements. Here we describe an ultrafast and accurate detector of somatic structural variations that reduces read-mapping costs by filtering out reads matched to pan-genome k-mer sets. The detector, which we named ETCHING (for efficient detection of chromosomal rearrangements and fusion genes), reduces the number of false positives by leveraging machine-learning classifiers trained with six breakend-related features (clipped-read count, split-reads count, supporting paired-end read count, average mapping quality, depth difference and total length of clipped bases). When benchmarked against six callers on reference cell-free DNA, validated biomarkers of structural variants, matched tumour and normal whole genomes, and tumour-only targeted sequencing datasets, ETCHING was 11-fold faster than the second-fastest structural-variant caller at comparable performance and memory use. The speed and accuracy of ETCHING may aid large-scale genome projects and facilitate practical implementations in precision medicine.

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development. The vascular cambium contains meristem cells that produce secondary xylem and phloem in the stems and roots of many plants. Its activity largely determines wood formation. Now, a genome-wide transcript profiling of Arabidopsis thaliana root cambium is presented to unlock the complex network that regulates cambium development and activity.

Purpose Targeted next-generation sequencing (NGS) panels for solid tumors have been useful in clinical framework for accurate tumor diagnosis and identifying essential molecular aberrations. However, most cancer panels have been designed to address a wide spectrum of pan-cancer models, lacking integral prognostic markers that are highly specific to gliomas. Materials and methods To address such challenges, we have developed a glioma-specific NGS panel, termed "GliomaSCAN," that is capable of capturing single nucleotide variations and insertion/deletion, copy number variation, and selected promoter mutations and structural variations that cover a subset of intron regions in 232 essential glioma-associated genes. We confirmed clinical concordance rate using pairwise comparison of the identified variants from whole exome sequencing (WES), immunohistochemical analysis, and fluorescence in situ hybridization. Results Our panel demonstrated high sensitivity in detecting potential genomic variants that were present in the standard materials. To ensure the accuracy of our targeted sequencing panel, we compared our targeted panel to WES. The comparison results demonstrated a high correlation. Furthermore, we evaluated clinical utility of our panel in 46 glioma patients to assess the detection capacity of potential actionable mutations. Thirty-two patients harbored at least one recurrent somatic mutation in clinically actionable gene. Conclusion We have established a glioma-specific cancer panel. GliomaSCAN highly excelled in capturing somatic variations in terms of both sensitivity and specificity and provided potential clinical implication in facilitating genome-based clinical trials. Our results could provide conceptual advance towards improving the response of genomically guided molecularly targeted therapy in glioma patients.

BACKGROUND: Prostate cancers exhibit intratumor heterogeneity (ITH), like other cancer types. The ITH may affect diverse phenotypes such as treatment response, drug resistance, and clinical outcomes. It is crucial to consider ITH to understand tumorigenesis. METHODS: Genomic and transcriptomic profiles of prostate cancer patients were investigated to determine which markers are correlated with the degree of tumor heterogeneity. In addition, the correlation between the immune activity and clonality of tumors was examined. RESULTS: Tumor heterogeneity across all prostate cancer samples was variable. However, ITH events were dependent on genomic and clinical features. Interestingly, prostate-specific antigen score increased in tumors with multiple subclones, indicating high-grade tumor heterogeneity. On the other hand, CD8-positive T-cell activation decreased in highly heterogeneous tumors. Intriguingly, PTEN deletion was prominently enriched in high heterogeneity groups, with a strong association with heterozygous loss. Expression of major genes including PTEN, CDC42EP5, RNLS, GP2, NETO2, and AMPD3 was closely related to tumor heterogeneity in association with PTEN deletion. CONCLUSIONS: In prostate cancer, ITH, a potential factor affecting tumor progression, is associated with PTEN deletion and cytotoxic T cell inactivation.

KGOG 3046 is a single-arm phase 2 study evaluating the combination of dual immune checkpoint inhibition (durvalumab [D] and tremelimumab [T]) with neoadjuvant chemotherapy (NAC) for the upfront advanced-stage epithelial ovarian cancer (aEOC). This study enrolled patients with FIGO stage IIIC-IV EOC. Patients were assigned to one of the following neoadjuvant chemo-immunotherapy (NACI): original cohort arm1 (D 1,500 mg q3w + T 75 mg q3w + paclitaxel 175 mg/m2 + carboplatin AUC 5 [3 cycles]) or expansion cohort arm2 (D 1,500 mg q3w + T 300 mg [1 dose] + same chemotherapy as original). Arm2 was initiated after the completion of arm1. After NAC, all patients underwent interval debulking surgery (IDS). After IDS, three cycles of D (1,120 mg) and adjuvant chemotherapy followed by D maintenance (1,120 mg [total 12 cycles]) were administered. During treatment, serial biopsies were performed to explore immunologic changes in tumor microenvironment (TME). The primary endpoint was a 12-month PFS rate. Interim analysis was performed to evaluate outcomes after NAC after all patients underwent IDS. A total of 45 patients were enrolled as follows: arm1 (n=23) and arm2 (n=22). The majority of the patients presented with high-grade serous carcinoma (91.1%) and stage IV disease (77.8%). After NAC, the ORR was 86.7% by RECIST v1.1 (95.7% in arm1 and 81.8% in arm2, P=0.17). At IDS, 30 patients (66.7%) had R0 resection (73.9% in arm1 and 59.1% in arm2, P=0.353). Fourteen patients (31.1%) had a CRS 3 (39.1% in arm1 and 22.7% in arm2, P=0.337) and 5 (11.1%) had a pathologic remission. Skin rashes were the most common adverse events (51.1%) and grade ≥3 events occurred in 3 patients (15.6%), which completely resolved after steroid use. Stromal TIL (P=0.0335), CD8 (P Summary of secondary outcomes D+T75+CT (n=23) D+T300+CT (n=22) P value ORR by RECIST 22 (95.7%) 18 (81.8%) 0.187 ORR by PERCIST 13/14a (92.9%) 16/18a (88.9%) 0.856 R0 at IDS 17 (73.9%) 13 (59.1%) 0.353 CRS3 at IDS 9 (39.1%) 5 (22.7%) 0.337 Pathologic CR 4 (17.4%) 1 (4.5%) 0.348 a, PERCIST was available in 14 (original) and 18 (expansion) cohorts. Citation Format: Junsik Park, Eunhyang Park, Je-Gun Joung, Myong Cheol Lim, Byoung-Gie Kim, Jae Weon Kim, Jung Bok Lee, Sunghoon Kim, Chel Hun Choi, Hee Seung Kim, Sang Yoon Park, Jongsuk Chung, Jung-Yun Lee. A phase II study of durvalumab and tremelimumab with front-line neoadjuvant chemotherapy in patients with advanced-stage ovarian cancer (KGOG 3046/TRU-D) [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 CT010.

PURPOSE: Relapsed/refractory pediatric cancers show poor prognosis; however, their genomic patterns remain unknown. To investigate the genetic mechanisms of tumor relapse and therapy resistance, we characterized genomic alterations in diagnostic and relapsed lesions in patients with relapsed/refractory pediatric solid tumors using targeted deep sequencing. PATIENTS AND METHODS: A targeted sequencing panel covering the exons of 381 cancer genes was used to characterize 19 paired diagnostic and relapsed samples from patients with relapsed/refractory pediatric solid tumors. RESULTS: The mean coverage for all samples was 930.6× (SD = 213.8). Among the 381 genes, 173 single nucleotide variations (SNVs)/insertion-deletions (InDels), 100 copy number alterations, and 1 structural variation were detected. A total of 72.6% of SNVs in primary tumors were also found in recurrent lesions, and 27.2% of SNVs in recurrent tumors had newly occurred. Among SNVs/InDels detected only in recurrent lesions, 71% had a low variant allele fraction (

Objectives: We hypothesized that adding durvalumab and tremelimumab to chemotherapy in advanced-stage epithelial ovarian cancer (aEOC) would increase progression-free survival (PFS) with minimal effects on safety. KGOG 3046 (NCT03899610) is a single-arm phase 2 study evaluating the combination of dual immune checkpoint inhibition and neoadjuvant chemotherapy (NAC) for the upfront treatment of aEOC. Methods: Patients with FIGO stage IIIC-IV EOC were offered three cycles of durvalumab (1500 mg), tremelimumab (75 mg) with chemotherapy for NAC followed by interval debulking surgery (IDS). After surgery, three cycles of durvalumab (1120 mg) and adjuvant chemotherapy followed by durvalumab maintenance (1120 mg [total 12 cycles]) were administered. During treatment, serial biopsies were performed at pre-treatment, IDS, and progression to identify immune biomarkers and changes in the tumor microenvironment. The primary endpoint was a 12 months PFS rate. Interim analysis was performed to evaluate outcomes after NAC (RECIST after NAC, R0 rate at IDS, the rate of CRS 3 at IDS, safety, a range of translational parameters) after all patients underwent IDS. Results: A total of 23 patients were enrolled with a median age of 60 years (range:44-77 years). The majority were presented with high-grade serous carcinoma (87.0%) and stage IV disease (60.9%). After NAC, 3 patients experienced a complete response (13.0%) and 20 patients had a partial response (87.0%). At IDS, R0 resection was achieved in 17 patients (73.9%); 9 patients (39.1%) had a CRS of 3; pathologic complete remission was achieved in 4 patients (17.4%). A total of 2 patients (8.7%) delayed IDS due to grade 4 skin rash and pneumonitis, respectively. Skin rashes were the most common adverse events (56.5%) and grade ≥3 events occurred in 3 patients (13.0%), which completely resolved after steroid use. Treatment was associated with tumor microenvironment conversion to an “inflamed” phenotype, with a significant increase in cytolytic index (P=0.015), stromal score (P Download : Download high-res image (81KB) Download : Download full-size image Conclusions: These interim data highlight the clinical activity and a manageable toxicity profile for the addition of durvalumab and tremelimumab to NAC in aEOC. Additional correlative data will be presented at the meeting. This study is actively enrolling patients in an expansion cohort.

Genome rearrangements often result in copy number alterations of cancer-related genes and cause the formation of cancer-related fusion genes. Current structural variation (SV) callers, however, still produce massive numbers of false positives (FPs) and require high computational costs. Here, we introduce an ultra-fast and high-performing somatic SV detector, called ETCHING, that significantly reduces the mapping cost by filtering reads matched to pan-genome and normal k-mer sets. To reduce the number of FPs, ETCHING takes advantage of a Random Forest classifier that utilizes six breakend-related features. We systematically benchmarked ETCHING with other SV callers on reference SV materials, validated SV biomarkers, tumor and matched-normal whole genomes, and tumor-only targeted sequencing datasets. For all datasets, our SV caller was much faster (≥15X) than other tools without compromising performance or memory use. Our approach would provide not only the fastest method for largescale genome projects but also an accurate clinically practical means for real-time precision medicine.

Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism that extends telomeres in cancer cells. It influences tumorigenesis and patient survival. Despite the clinical significance of ALT in tumors, the manner in which ALT is activated and influences prognostic outcomes in distinct cancer types is unclear. In this work, we profiled distinct telomere maintenance mechanisms (TMMs) using 8953 transcriptomes of 31 different cancer types from The Cancer Genome Atlas (TCGA). Our results demonstrated that approximately 29% of cancer types display high ALT activity with low telomerase activity in the telomere-lengthening group. Among the distinct ALT mechanisms, homologous recombination was frequently observed in sarcoma, adrenocortical carcinoma, and kidney chromophobe. Five cancer types showed a significant difference in survival in the presence of high ALT activity. Sarcoma patients with elevated ALT had unfavorable risks (p &lt, 0.038) coupled with a high expression of TOP2A, suggesting this as a potential drug target. On the contrary, glioblastoma patients had favorable risks (p &lt, 0.02), and showed low levels of antigen-presenting cells. Together, our analyses highlight cancer type-dependent TMM activities and ALT-associated genes as potential therapeutic targets.

Summary Exome and transcriptome analyses of clinically homogeneous early-stage never-smoker female patients with lung adenocarcinoma were performed to understand tumor-T cell interactions and immune escape points. Using our novel gene panels of eight functional categories in the cancer-immunity cycle, three distinct subgroups were identified in this immune checkpoint blockade-refractory cohort by defective gene expression in two major domains, i.e., type I interferon production/signaling pathway and antigen-presenting machinery. Our approach could play a critical role in understanding immune evasion mechanisms, developing a method for effective selection of rare immune checkpoint blockade responders, and finding new treatment strategies., Graphical Abstract, Highlights • GMM-based clustering of cancer-immune gene expression helps to find LUAD classifiers • Classifiers are found in the earlier-phased cancer-immunity cycle • Classifiers include ubiquitination, Ag-presenting machinery, and type I IFN signaling • Tumor-intrinsic classifiers strongly influence the outcome of cancer immunity, Immunology; Cancer Systems Biology; Genomics

Despite the increasing incidence of pathological stage T1 renal cell carcinoma (pT1 RCC), postoperative distant metastases develop in many surgically treated patients, causing death in certain cases. Therefore, this study aimed to create a radiomics model using imaging features from multiphase computed tomography (CT) to more accurately predict the postoperative metastasis of pT1 RCC and further investigate the possible link between radiomics parameters and gene expression profiles generated by whole transcriptome sequencing (WTS). Four radiomic features, including the minimum value of a histogram feature from inner regions of interest (ROIs) (INNER_Min_hist), the histogram of the energy feature from outer ROIs (OUTER_Energy_Hist), the maximum probability of gray-level co-occurrence matrix (GLCM) feature from inner ROIs (INNER_MaxProb_GLCM), and the ratio of voxels under 80 Hounsfield units (Hus) in the nephrographic phase of postcontrast CT (Under80HURatio), were detected to predict the postsurgical metastasis of patients with pathological stage T1 RCC, and the clinical outcomes of patients could be successfully stratified based on their radiomic risk scores. Furthermore, we identified heterogenous-trait-associated gene signatures correlated with these four radiomic features, which captured clinically relevant molecular pathways, tumor immune microenvironment, and potential treatment strategies. Our results of accurate surrogates using radiogenomics could lead to additional benefit from adjuvant therapy or postsurgical metastases in pT1 RCC.

Purpose Neuroblastoma (NB) is the most common extracranial solid tumor found in children. To identify significant genetic factors for the risk of NB, several genetic studies was conducted mainly for Caucasians and Europeans. However, considering racial differences, there is a possibility that genetic predispositions that contribute to the development of NB are different, and genome-wide association study has not yet been conducted on Korean NB patients. Materials and Methods To identify the genetic variations associated with the risk of pediatric NB in Korean children, we performed a genome-wide association analysis with 296 NB patients and 1,000 unaffected controls (total n=1,296) after data cleaning and filtering as well as imputation of non-genotyped single nucleotide polymorphisms (SNPs) using IMPUTE v2.3.2. Results After adjusting for multiple comparisons, we found 21 statistically significant SNPs associated with the risk of NB (Pcorr < 0.05) within 12 genes (RPTN, MRPS18B, LRRC45, KANSL1L, ARHGEF40, IL15RA, L1TD1, ANO7, LAMA5, OR7G2, SALL4, and NEUROG2). Interestingly, out of these, 12 markers were nonsynonymous SNPs. The SNP rs76015112 was most significantly associated with the risk of NB (p = 8.1E-23, Pcorr = 2.3E-17) and was located in the RPTN gene. In addition, significant nonsynonymous SNPs in ADGRE1 were found in patients with MYCN amplification (rs7256147, p = 2.6E-05). In high-risk group, rs7256147 was observed as a significant SNP (p = 5.9E-06). Conclusion Our findings might facilitate improved understanding of the mechanism of pediatric NB pathogenesis. However, functional evaluation and replication of these results in other populations are still needed.

BACKGROUND: Signet-ring cell carcinoma (SRCC) is a very rare subtype of colorectal adenocarcinoma (COAD) with a poor clinical prognosis. Although understanding key mechanisms of tumor progression in SRCCs is critical for precise treatment, a comprehensive view of genomic alterations is lacking. MATERIALS AND METHODS: We performed whole-exome sequencing of tumors and matched normal blood as well as RNA sequencing of tumors and matched normal colonic tissues from five patients with SRCC. RESULTS: We identified major somatic alterations and characterized transcriptional changes at the gene and pathway level. Based on high-throughput sequencing, the pattern of mutations and copy number variations was overall similar to that of COAD. Transcriptome analysis revealed that major transcription factors, such as SRF, HNF4A, ZEB1, and RUNX1, with potential regulatory roles in key pathways, including focal adhesion, the PI3K-Akt signaling pathway, and the MAPK signaling pathway, may play a role in the tumorigenesis of SRCC. Furthermore, significantly upregulated genes in SRCCs were enriched for epithelial-mesenchymal transition genes, and accumulation of mucin in intracytoplasm was associated with the overexpression of MUC2. CONCLUSION: The results indicate that the molecular basis of colorectal SRCC exhibits key differences from that of consensus COAD. Our findings clarify important genetic features of particular abnormalities in SRCCs.

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.

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.

Polo-like kinase 1 (PLK1) is a critical regulator of cell cycle progression and apoptosis. However, its regulation remains poorly understood. In the present study, we investigated the molecular mechanism underlying the post-transcriptional regulation of PLK1. We observed that heterogeneous nuclear ribonucleoprotein K (hnRNPK) and PLK1 were positively associated in several different cancers and high expression levels of them correlated with poor prognosis in patients with cancer. Knockdown of hnRNPK resulted in reduced expression of PLK1, whereas conversely, PLK1 expression was increased in hnRNPK-overexpressing cells. We found that hnRNPK regulated PLK1 expression through KH1- and KH2-dependent interactions with the 3′UTR of PLK1 mRNA. In addition, microRNA-149-3p (miR-149-3p) and miR-193b-5p suppressed PLK1 expression by targeting the 3′UTR of PLK1 mRNA. MicroRNA-elicited enrichment of PLK1 mRNA in Ago2 immunoprecipitation was altered by the presence or absence of hnRNPK. Furthermore, the deletion of the cytosine (C)-rich sequences of the 3′UTR of PLK1 mRNA abolished the decreased PLK1 expression observed via hnRNPK silencing and administration of miRNAs, a finding that suggests that hnRNPK shares this C-rich motif with miR-149-3p and miR-193b-5p. We also found that downregulation of PLK1 by either silencing hnRNPK or overexpression of miR-149-3p and miR-193b-5p decreased clonogenicity and induced apoptosis. Our findings from this study demonstrate that hnRNPK regulates PLK1 expression by competing with the PLK1-targeting miRNAs, miR-149-3p and miR-193b-5p.

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.

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 The immunogenomic changes triggered by concurrent chemoradiation therapy (CCRT), a standard neoadjuvant treatment for locally advanced esophageal squamous cell carcinoma (ESCC), are unknown. We aimed to analyze the early immunogenomic changes in ESCC induced by CCRT and to correlate them with clinical outcomes. Methods We collected biopsy samples from 40 patients with ESCC and the surgical candidates were treated with 5-fluorouracil (5-FU)/Cisplatin and concurrent radiation therapy. Endoscopic biopsy was performed before and after one treatment cycle of 5-FU/Cisplatin and 5 to 18 fractions of radiation. We analyzed immunogenomic changes using paired whole-exome sequencing (n = 29) and paired whole-transcriptome sequencing (WTS, n = 23). Multiplex immunohistochemistry (IHC) was conducted in four representative pair samples. Results Fourteen out of 23 WTS samples (60.8%) showed increased immune scores after CCRT, as calculated by ESTIMATE. The rate of progression-free survival was higher in patients with increased immune scores compared with the remaining patients (83.1% vs. 57.1%, p = 0.25). Tumor mutation burden and neoantigen load were significantly reduced after CCRT (p

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity

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.

Figure S1. Heatmap comparison of differentially expressed genes between pre- and post-concurrent chemoradiation in the non-pathologic complete response sample group. (PDF 499 kb)

Figure S2. (a) comparison between samples from the patients who showed disease progression (PD) to neo-adjuvant chemotherapy (n = 2) and the samples from the patients (Other) who received surgery (n = 25). (b) Specific immune cells which showed significant higher fraction compared between the neoadjuvant chemotherapy PD patients and other patients (PDF 543 kb)

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.

// Daeun Ryu 1, 4, * , Hee Jin Kim 2, * , Je-Gun Joung 1, * , Hae-Ock Lee 1, 5 , Joon Seol Bae 1 , Seok Jin Kim 3 , Haesu Kim 3 , Woong-Yang Park 1, 4, 5 , Kihyun Kim 3 1 Samsung Genome Institute, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Department of Laboratory Medicine & Genetics, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 4 Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, Korea 5 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Kihyun Kim, email: kihyun.kim@samsung.com Woong-Yang Park, email: woongyang.park@samsung.com Keywords: multiple myeloma, IgM, IRF4, progression-free survival, sequencing Received: October 16, 2015 Accepted: March 28, 2016 Published: May 19, 2016 ABSTRACT Immunoglobulin M multiple myeloma (IgM MM) is an extremely rare subtype of multiple myeloma with a poor clinical outcome. In this study, bone marrow aspirates of MM patients, including two cases of IgM MM, were analyzed by whole exome sequencing and RNA sequencing. Recurrent somatic mutations in the NRAS , KRAS , CCND1 , DIS3 , and TP53 genes were found in IgM MM and other types of MM, in agreement with previous studies. Overall transcription profiles of IgM and other types of MM clustered together, but separate from normal blood or peripheral plasma cells. Among the differentially expressed genes in IgM MM, IRF4 was highly expressed in IgM as well as in a subset of other types of MM patients. Thus, IRF4 is an independent prognostic factor for general MM patients. Taken together, the somatic mutation and transcriptome profiles support the idea that IgM MM can be classified as an aggressive MM subtype.

Intratumor heterogeneity within individual cancer tissues underlies the numerous phenotypes of cancer. Tumor subclones ultimately affect therapeutic outcomes due to their distinct molecular features. Drug-resistant subclones are present at a low frequency in tissues at the time of biopsy, but can also arise as a result of acquired somatic mutations. A number of different approaches have been utilized to understand the nature of intratumor heterogeneity. Clonal analysis using whole exome or genome sequencing data can help monitor subclones in the context of tumor progression. Multiregional biopsies permit the molecular characterization of subclones within tumors. Deep sequencing has also provided researchers with the ability to measure the low allele fraction variant within a small number of cells. Ultimately, single-cell sequencing will enable the identification of every minor population within a tumor microenvironment. In the clinical context, the ability to identify and monitor the subclonal architecture of a tumor is valuable for the development of precise cancer therapeutic methods.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor derived from non-neuronal glial cells. Neurofibromatosis 2 (NF2) protein, also termed as merlin, is a well-known tumor suppressor; however, the molecular mechanism underlying this effect has not yet been fully defined. To investigate the role of NF2 in the invasiveness of GBM, we used two GBM cell lines: NF2-expressing T98G cells and NF2-deficient A172 cells. Knockdown of NF2 increased the invasiveness of T98G cells, whereas NF2-overexpressing A172 cells showed decreased invasive activity. Moreover, re-expression of NF2 reversed the high invasiveness of NF2-silenced T98G cells, indicating that NF2 negatively regulates GBM invasiveness. We further found that the NF2-mediated regulation of invasiveness was dependent on YAP and TEAD2 expression levels. NF2 also controlled the expression of YAP targets, including cysteine-rich angiogenic inducer 61 (CYR61/CCN1), by regulating the nuclear localization of YAP. Silencing of CYR61/CCN1 blocked the increased invasiveness of T98G cells, suggesting that CYR61/CCN1 is required for NF2-mediated invasiveness. Through microRNA microarray analysis, we found that NF2 negatively regulates the expression of miR-296-3p. Overexpression of miR-296-3p suppressed the expression of STAT5A, induced the phosphorylation of STAT3 by downregulating SOCS2, and increased the invasiveness of T98G cells. Taken together, we demonstrate that NF2 negatively controls the invasiveness of GBM through YAP-dependent induction of CYR61/CCN1 and miR-296-3p.

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

This study investigated changes in the intestinal microbiota during 8-week infliximab maintenance therapy in inflammatory bowel disease (IBD) patients in clinical remission. Microbial compositional differences were analyzed according to the trough level of infliximab (TLI) and mucosal healing (MH) status. 16S rRNA gene-based microbiome profiling was performed on 10 and 74 fecal samples from 10 healthy volunteers and 40 adult IBD patients, respectively. Fecal sampling occurred at 1&ndash, 2 weeks (1W) and 7&ndash, 8 weeks (7W) after infliximab infusion. TLI was measured by ELISA at 8 weeks, immediately before the subsequent infusion, MH was evaluated by endoscopy within 3 months. There were no significant changes in microbial composition, species richness, or diversity indices between 1W and 7W. However, 7W samples from the patients with TLI &ge, 5 &mu, g/mL showed an increased species richness compared with patients with TLI &lt, g/mL, and patients with MH showed increased diversity compared with non-MH patients. Beta-diversity analysis showed clustering between samples in the MH and non-MH groups. LEfSe analysis identified differential composition of Faecalibacterium prausnitzii group according to TLI and MH. In conclusion, these results suggest the potential of fecal microbiota as a response indicator.

In-depth molecular pathogenesis of ground-glass nodular lung adenocarcinoma has not been well understood. The objectives of this study were to identify genomic alterations in ground-glass nodular lung adenocarcinomas and to investigate whether viral transcripts were detected in these tumors. Nine patients with pure (n = 4) and part-solid (n = 5) ground-glass nodular adenocarcinomas were included. Six were females with a median age of 58 years. We performed targeted exon sequencing and RNA sequencing. EGFR (n = 10), IDH2 (n = 2), TP53 (n = 1), PTEN (n = 1), EPHB4 (n = 1), and BRAF (n = 1) were identified as driver mutations by targeted exon sequencing. Vasculogenesis-associated genes including NOTCH4 and TGFBR3 expression were significantly downregulated in adenocarcinoma tissue versus normal tissue (adjusted P values

This study investigated the correlations between parameters of 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET) scan and indices of genetic properties, heterogeneity index (HI), and tumor mutation burden (TMB), in patients with lung cancer. We produced 106 PET indices for each tumor site that underwent genomic analysis in a total of 176 study subjects (age, 62.0 ± 10.0 y; males, 68.2%), comprising 101 adenocarcinoma (ADC), 29 squamous cell carcinoma (SQCC), and 46 small cell lung cancer (SCLC) patients. We then examined the correlations of the PET parameters with genetic properties of HI and TMB, according to pathology and tumor site. Comparisons between PET parameters and the genetic properties with false discovery rate (FDR) correction revealed that the surface standard uptake value (SUV) entropy of SUV statistics had a significant correlation with HI only in patients with SCLC who underwent a genetic test in lymph nodes (r = 0.592, p = 0.028), whereas PET parameters did not show a significant correlation with HI or TMB in patients with SCLC who underwent a genetic test in lung tissue. In patients with ADC and SQCC, there was no significant correlation between PET parameters and the genetic properties. Although SUVmax showed raw p values less than 0.05 in correlation with HI (r = 0.315, raw p = 0.048) and TMB (r = 0.206, raw p = 0.043) in ADC, and SUVpeak had a raw p value less than 0.05 in correlation with HI (r = 0.394, raw p = 0.046) in SQCC, these parameters were not significant when corrected by FDR. In this study, surface SUV entropy had a significant correlation with HI in SCLC. Regarding other PET parameters and tumors, no significant correlation with genetic parameters existed.