Search

Bo Young Oh,1,* Jeonghee Cho,2,* Hye Kyung Hong,3 Joon Seol Bae,4 Woong-Yang Park,4–6 Je-Gun Joung,4 Yong Beom Cho3,5 1Department of Surgery, College of Medicine, Ewha Womans University, Seoul, 2Department of Nanobiomedical Science, Dankook University, Cheonan, 3Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 4Samsung Genome Institute, Samsung Medical Center, 5Department of Health Sciences and Technology, Samsung Advanced Institute for Health Science and Technology, Sungkyunkwan University, 6Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea *These authors contributed equally to this work 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

Objective: Effective functional biomarkers that can be readily used in clinical practice to predict poly(ADP-ribose) polymerase inhibitor (PARPi) sensitivity are lacking. With the widespread adoption of PARPi maintenance therapy in ovarian cancer, particularly in patients with BRCA mutation or HR deficiencies, accurately identifying de novo or acquired resistance to PARPi has become critical in clinical practice. We investigated RAD51 immunohistochemistry (IHC) as a functional biomarker for predicting PARPi sensitivity in ovarian cancer., Methods: Ovarian cancer patients who had received PARPi and had archival tissue samples prior to PARPi exposure ("pre-PARPi") and/or after progression on PARPi ("post-PARPi") were selected. RAD51 IHC expression was semi-quantitatively evaluated using the H-score in geminin (a G2/S phase marker)- and γH2AX (a DNA damage marker)-positive tissues. A RAD51 H-score of 20 was used as the cutoff value., Results: In total, 72 samples from 56 patients were analyzed. The median RAD51 H-score was 20 (range: 0-90) overall, 10 (0-190) in pre-PARPi samples (n = 34), and 25 (1-170) in post-PARPi samples (n = 19). Among patients with BRCA mutations, RAD51-low patients had better progression-free survival (PFS) after PARPi treatment than RAD51-high patients ( P = 0.029). No difference was found in PFS with respect to the genomic scar score ( P = 0.930). Analysis of matched pre- and post-PARPi samples collected from 15 patients indicated an increase in the RAD51 H-score upon progression on PARPi, particularly among pre-PARPi low-RAD51-expressing patients., Conclusion: RAD51 is a potential functional IHC biomarker of de novo and acquired PARPi resistance in BRCA -mutated ovarian cancer and can be used to fine-tune ovarian cancer treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Kim, Joung, Kim, Kim, Lee and Park.)

Aim: We investigated the efficacy and safety of durvalumab (D) with or without tremelimumab (T) in addition to single-agent chemotherapy (CT) in patients with platinum-resistant recurrent ovarian cancer (PROC) lacking homologous recombination repair (HRR) gene mutations., Patients and Methods: KGOG 3045 was an open-label, investigator-initiated phase II umbrella trial. Patients with PROC without HRR gene mutations who had received ≥2 prior lines of therapy were enrolled. Patients with high PD-L1 expression (TPS ≥25%) were assigned to arm A (D + CT), whereas those with low PD-L1 expression were assigned to arm B (D + T75 + CT). After completing arm B recruitment, patients were sequentially assigned to arms C (D + T300 + CT) and D (D + CT)., Results: Overall, 58 patients were enrolled (5, 18, 17, and 18 patients in arms A, B, C, and D, respectively). The objective response rates were 20.0, 33.3, 29.4, and 22.2%, respectively. Grade 3-4 treatment-related adverse events were observed in 20.0, 66.7, 47.1, and 66.7 of patients, respectively, but were effectively managed. Multivariable analysis demonstrated that adding T to D + CT improved progression-free survival (adjusted HR, 0.435; 95% CI, 0.229-0.824; P = 0.011). Favorable response to chemoimmunotherapy was associated with MUC16 mutation (P = 0.0214), high EPCAM expression (P = 0.020), high matrix remodeling gene signature score (P = 0.017), and low FOXP3 expression (P = 0.047). Patients showing favorable responses to D + T + CT exhibited significantly higher EPCAM expression levels (P = 0.008) and matrix remodeling gene signature scores (P = 0.031) than those receiving D + CT., Conclusions: Dual immunotherapy with chemotherapy showed acceptable response rates and tolerable safety in HRR non-mutated PROC, warranting continued clinical investigation., Competing Interests: Declaration of competing interest JYL, BGK, and JWK disclose financial support from AstraZeneca during the study. JYL reports grants and personal fees from multiple companies, including Beigene, Bergenbio, Clovis Oncology, Immunogen, Janssen, Merck, MSD, Novartis, Roche, Seagen, Synthon, and Takeda. BGK reports grants from MSD, Cellid, and Utilex. JWK reports personal fees from AstraZeneca, Janssen, MSD, Takeda, GSK, Boryung, CMIC, LG Pharma, and Viflor Pharma. All other authors (SIK, JGJ, YNK, JP, EP, JBL, SK, CHC, HSK, JL, and JC) have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Choosing an optimal concomitant drug for combination with poly-ADP ribose polymerase (PARP) inhibitor based on patient-specific biomarker status may help increase to improve treatment efficacy in patients with ovarian cancer. However, the efficacy and safety of different PARP inhibitor-based combinations in patients with homologous recombination repair (HRR) mutations have not been evaluated in ovarian cancer. In this sub-study of Korean Gynecologic Oncology Group (KGOG) 3045, we compared the efficacy and safety of two olaparib-based combinations and biomarkers of patients with platinum-resistant ovarian cancer with HRR gene mutations. Patients were randomized to receive either olaparib (200 mg twice a day) + cediranib (30 mg daily) (Arm 1, n = 16) or olaparib (300 mg) + durvalumab (1,500 mg once every 4 weeks) (Arm 2, n = 14). The objective response rates for Arm 1 and Arm 2 were 50.0% and 42.9%, respectively. Most patients (83.3%) had BRCA mutations, which were similarly distributed between arms. Grade 3 or 4 treatment-related adverse events were observed in 37.5% and 35.7% of the patients, respectively, but all were managed properly. A high vascular endothelial growth factor signature was associated with favorable outcomes in Arm 1, whereas immune markers (PD-L1 expression [CPS ≥10], CD8, neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio) were associated with favorable outcomes in Arm 2. The activation of homologous recombination pathway upon disease progression was associated with poor response to subsequent therapy. Based on comprehensive biomarker profiling, including immunohistochemistry, whole-exome and RNA sequencing and whole blood-based analyses, we identified biomarkers that could help inform which of the two combination strategies is appropriate given a patient's biomarker status. Our findings have the potential to improve treatment outcome for patients with ovarian cancer in the PARP inhibitor era., (© 2023 UICC.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

High-grade serous ovarian carcinoma (HGSOC) is a fatal gynecological malignancy. Somatic recombination occurring during T-cell receptor (TCR) development results in TCR diversity, and the TCR repertoire, thus produced, is associated with immune response. This study analyzed the difference in the TCR repertoire and their prognostic significance in 51 patients with HGSOC. The patient's clinical characteristics, gene expression pattern, TCR clonotypes, and degree of tumor-infiltrating leukocytes (TILs) were analyzed, and the patients were divided into groups depending on their recurrence pattern, tumor-infiltrating leukocyte (TIL) score, and homologous recombinant repair pathway deficiency (HRD)-associated mutations. The TCR repertoire was low in patients with recurrence and showed the expansion of eight TCR segments. Interestingly, a few genes correlated with the TCRs also showed a difference in expression according to the prognosis. Among them, seven genes were related to immune responses and KIAA1199 was up-regulated in ovarian cancer. Our study shows that the differences in the TCR repertoire in patients with ovarian cancer and their associated immune pathways could affect the prognosis of HGSOC.

Background: Recurrent glioblastoma multiforme (GBM) is a highly aggressive primary malignant brain tumor that is resistant to existing treatments. Recently, we reported that activated autologous natural killer (NK) cell therapeutics induced a marked increase in survival of some patients with recurrent GBM., Methods: To identify biomarkers that predict responsiveness to NK cell therapeutics, we examined immune profiles in tumor tissues using NanoString nCounter analysis and compared the profiles between 5 responders and 7 non-responders. Through a three-step data analysis, we identified three candidate biomarkers (TNFRSF18, TNFSF4, and IL12RB2) and performed validation with qRT-PCR. We also performed immunohistochemistry and a NK cell migration assay to assess the function of these genes., Results: Responders had higher expression of many immune-signaling genes compared with non-responders, which suggests an immune-active tumor microenvironment in responders. The random forest model that identified TNFRSF18, TNFSF4, and IL12RB2 showed a 100% accuracy (95% CI 73.5-100%) for predicting the response to NK cell therapeutics. The expression levels of these three genes by qRT-PCR were highly correlated with the NanoString levels, with high Pearson's correlation coefficients (0.419 (TNFRSF18), 0.700 (TNFSF4), and 0.502 (IL12RB2)); their prediction performance also showed 100% accuracy (95% CI 73.54-100%) by logistic regression modeling. We also demonstrated that these genes were related to cytotoxic T cell infiltration and NK cell migration in the tumor microenvironment., Conclusion: We identified TNFRSF18, TNFSF4, and IL12RB2 as biomarkers that predict response to NK cell therapeutics in recurrent GBM, which might provide a new treatment strategy for this highly aggressive tumor., (© 2023. The Author(s).)

The risk of chromosomal abnormalities in the child increases with increasing maternal age. Although non-invasive prenatal testing (NIPT) is a safe and effective prenatal screening method, the accuracy of the test results needs to be improved owing to various testing conditions. We attempted to achieve a more accurate and robust prediction of chromosomal abnormalities by combining multiple methods. Here, three different methods, namely standard Z-score, normalized chromosome value, and within-sample reference bin, were used for 1698 reference and 109 test samples of whole-genome sequencing. The logistic regression model combining the three methods achieved a higher accuracy than any single method. In conclusion, the proposed method offers a promising approach for increasing the reliability of NIPT.

Purpose: In early-stage, EGFR mutation-positive (EGFR-M+) non-small cell lung cancer (NSCLC), surgery remains the primary treatment, without personalized adjuvant treatments. We aimed to identify risk factors for recurrence-free survival (RFS) to suggest personalized adjuvant strategies in resected early-stage EGFR-M+ NSCLC., Experimental Design: From January 2008 to August 2020, a total of 2,340 patients with pathologic stage (pStage) IB-IIIA, non-squamous NSCLC underwent curative surgery. To identify clinicopathologic risk factors, 1,181 patients with pStage IB-IIIA, common EGFR-M+ NSCLC who underwent surgical resection were analyzed. To identify molecular risk factors, comprehensive genomic analysis was conducted in 56 patients with matched case-controls (pStage II and IIIA and type of EGFR mutation)., Results: Median follow-up duration was 38.8 months (0.5-156.2). Among 1,181 patients, pStage IB, II, and IIIA comprised 577 (48.9%), 331 (28.0%), and 273 (23.1%) subjects, respectively. Median RFS was 73.5 months [95% confidence interval (CI), 62.1-84.9], 48.7 months (95% CI, 41.2-56.3), and 22.7 months (95% CI, 19.4-26.0) for pStage IB, II, and IIIA, respectively (P < 0.001). In multivariate analysis of clinicopathologic risk factors, pStage, micropapillary subtype, vascular invasion, and pleural invasion, and pathologic classification by cell of origin (type II pneumocyte-like tumor cell vs. bronchial surface epithelial cell-like tumor cell) were associated with RFS. As molecular risk factors, the non-terminal respiratory unit (non-TRU) of the RNA subtype (HR, 3.49; 95% CI, 1.72-7.09; P < 0.01) and TP53 mutation (HR, 2.50; 95% CI, 1.24-5.04; P = 0.01) were associated with poor RFS independent of pStage II or IIIA. Among the patients with recurrence, progression-free survival of EGFR-tyrosine kinase inhibitor (TKI) in those with the Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) mutation signature was inferior compared with that of patients without this signature (8.6 vs. 28.8 months; HR, 4.16; 95% CI, 1.28-13.46; P = 0.02)., Conclusions: The low-risk group with TRU subtype and TP53 wild-type without clinicopathologic risk factors might not need adjuvant EGFR-TKIs. In the high-risk group, with non-TRU subtype and/or TP 53 mutation, or clinicopathologic risk factors, a novel adjuvant strategy of EGFR-TKI with others, e.g., chemotherapy or antiangiogenic agents needs to be investigated. Given the poor outcome to EGFR-TKIs after recurrence in patients with the APOBEC mutation signature, an alternative adjuvant strategy might be needed., (©2022 American Association for Cancer Research.)

Background: The intestinal microenvironment directly determines the human T-cell receptor (TCR) repertoire. Despite its extreme diversity, TCR repertoire analysis may provide a better understanding of the immune system in patients with inflammatory bowel disease., Methods: To investigate TCR repertoires in the intestinal mucosa, RNA sequencing was performed for inflamed and non-inflamed intestinal mucosa samples obtained from 13 patients with Crohn's disease (CD) and healthy mucosa from nine non-IBD controls., Results: The gene expression frequency of the TCR repertoire showed a clear separation between inflamed mucosa of patients with CD and healthy mucosa of non-IBD controls in the hierarchical clustering heatmap. The richness of TCR repertoires measured by the Chao1 index did not show a significant difference among groups, whereas diversity measured by the D50 diversity index was decreased in the inflamed mucosa of CD patients. Rare/small TCR clonotypes occupied a large proportion of TCR repertoires in healthy mucosa of controls, whereas expanded clonotypes were common in inflamed mucosa of patients with CD. Segment usages of TRAV2, TRAV22, TRAV40, TRJ14, TRAJ51, TRBV1, TRBV21.1, and TRBJ1.5 were significantly decreased in CD patients. KEGG enrichment analysis identified the enrichment of several KEGG pathways, including inflammatory bowel disease ( p = 0.0012), Th1 and Th2 cell differentiation ( p = 0.0011), and intestinal immune network for IgA production ( p = 0.0468)., Conclusions: The diversity of the TCR repertoire is reduced in inflamed mucosa of CD patients, which might contribute to intestinal inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hong, Park, Yang, Lee, Kim and Joung.)

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 germline telomere length. The germline telomere length of peripheral blood mononuclear cells from 186 patients with neuroblastoma was measured by quantitative polymerase chain reaction. 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 bead chip (Illumina, San Diego, CA, USA). After strict quality-control checks of the samples, an association analysis was conducted. The result showed that longer germline telomeres were significantly associated with longer event-free survival (P = 0.032). To identify significantly assocated genetic markers for germline telomere length, genome wide association analysis was performed. As a result, several single nucleotide polymorphisms located in HIVEP3, LRRTM4, ADGRV1, RAB30, and CHRNA4 genes were discovered. During gene-based analysis (VEGAS2 tool), the CNTN4 gene had the most significant association with germline telomere length (P = 1.0E-06). During gene ontology analysis, susceptible genes associated with germline telomere length were mainly distributed in neurite morphogenesis and neuron development. A 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 germline telomere length are associated with neurite morphogenesis and neuron development. Further research with larger cohorts of patients and functional investigations are needed., (© 2022. The Author(s).)

Objective: 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., Methods: 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)., Results: 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., Conclusion: 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., Trial Registration: ClinicalTrials.gov Identifier: NCT03699449., Competing Interests: JYL reports grants from AstraZeneca during the conduct of the study; grants and personal fees from Beigene, Bergenbio, Clovis Oncology, Immunogen, Janssen, Merck, MSD, Novartis, Roche, Seagen, Synthon, and Takeda. All other authors have no conflicts of interest to disclose., (Copyright © 2022. Asian Society of Gynecologic Oncology, Korean Society of Gynecologic Oncology, and Japan Society of Gynecologic Oncology.)

The dynamic changes in the tumor immune microenvironment (TIME) triggered by neoadjuvant chemotherapy (NAC) have not been clearly defined in advanced-stage ovarian cancer. We analyzed the immunologic changes induced by NAC to correlate them with clinical outcomes. We compared the changes in the immune infiltration of high-grade serous carcinoma biopsies before and after NAC via immunohistochemistry (147 paired samples) and whole transcriptome sequencing (35 paired samples). Immunohistochemistry showed significantly increased PD-L1 levels and TIL levels after NAC. Whole transcriptome sequencing revealed that the stromal score, immune score, and cytolytic activity score significantly increased after NAC. An increased tumor-infiltrating lymphocyte (TIL) level in response to NAC was associated with shorter progression-free survival compared with decreased TIL level after NAC. In tumors with increased TIL levels after NAC, the relative fraction of CD8 T cells and regulatory T cells significantly increased with immunohistochemistry. Post-NAC tumors were enriched in gene sets associated with immune signaling pathways, such as regulatory T cell and JAK/STAT signaling pathways. NAC induced dynamic changes in the TIME that increased TIL levels, but their high abundance did not impart any survival benefit. Our data may provide therapeutic strategies to improve the survival benefit from immunotherapies in ovarian cancer.

Introduction: The prognostic value of F-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) in hepatocellular carcinoma (HCC) was established in previous reports. However, there is no evidence suggesting the prognostic value of transcriptomes associated with tumor FDG uptake in HCC. It was aimed to elucidate metabolic genes and functions associated with FDG uptake, followed by assessment of those prognostic value., Methods: Sixty HCC patients with Edmondson-Steiner grade II were included. FDG PET/CT scans were performed before any treatment. RNA sequencing data were obtained from tumor and normal liver tissue. Associations between each metabolism-associated gene and tumor FDG uptake were investigated by Pearson correlation analyses. A novel score between glucose and lipid metabolism-associated gene expression was calculated. In The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset, the prognostic power of selected metabolism-associated genes and a novel score was evaluated for external validation., Results: Nine genes related to glycolysis and the HIF-1 signaling pathway showed positive correlations with tumor FDG uptake; 21 genes related to fatty acid metabolism and the PPAR signaling pathway demonstrated negative correlations. Seven potential biomarker genes, PFKFB4 , ALDOA , EGLN3 , EHHADH , GAPDH , HMGCS2 , and ENO2 were identified. A metabolic gene expression balance score according to the dominance between glucose and lipid metabolism demonstrated good prognostic value in HCC., Conclusions: The transcriptomic evidence of this study strongly supports the prognostic power of FDG PET/CT and indicates the potential usefulness of FDG PET/CT imaging biomarkers to select appropriate patients for metabolism-targeted therapy in HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Choi, Joung, Joh, Kim and Hyun.)

Background & Aims: Tumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn's disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids., Methods: Human enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated., Results: TNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing., Conclusions: TNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Young patients with colorectal cancer (CRC) exhibit poor prognoses compared to older patients due to the difficulty in early diagnosis and treatment. However, the underlying molecular characteristics are still unclear., Methods: We conducted a comprehensive analysis of 49 CRC patients without hereditary CRC using the whole-exome and RNA sequencing with tumor and matched normal samples. A total of 594 TCGA samples and 4 patient-derived cells were utilized for validation., Results: Consensus molecular subtype 4 (CMS4) (53.85%) and CMS2 (38.46%) were enriched in the young (≤ 40 years) and old (> 60 years) age groups, respectively. A CMS4-associated gene, platelet-derived growth factor receptor α (PDGFRA), was significantly upregulated in young patients with CRC (FC = 3.21, p = 0.0001) and was negatively correlated with age (p = 0.0001, R = - 0.526). Moreover, PDGFRA showed a positive co-expression with metastasis-related genes in young CRC patients. In vitro validation confirmed that young patient-derived cells (PDCs) showed an enriched expression of PDGFRA compared to old PDCs and a reduced proliferation rate by knockdown of PDGFRA. Furthermore, young CRC patients were more sensitive to regorafenib, a PDGFRA-targeting drug, than old CRC patients., Conclusions: Our study suggests that CRC in young patients is associated with CMS4 and PDGFRA. In addition, PDGFRA may serve potential of novel therapeutic strategies and represent a predictive biomarker of response to regorafenib for young CRC patients., (© 2021. The Author(s).)

Previous studies suggested that genetic, environmental factors and their interactions could affect body fat mass (BFM). However, studies describing these effects were performed at a single time point in a population. In this study, we investigated the interaction between genetic and environmental factors in affecting BFM and implicate the healthcare utilization of lifestyle modifications from a personalized and genomic perspective. We examined how nutritional intake or physical activity changes in the individuals affect BFM concerning the genetic composition. We conducted an observational study including 259 adult participants with single nucleotide polymorphism (SNP) genotyping and longitudinal lifestyle monitoring, including food consumption and physical activities, by following lifestyle modification guidance. The participants' lifelog data on exercise and diet were collected through a wearable device for 3 months. Moreover, we measured anthropometric and serologic markers to monitor their potential changes through lifestyle modification. We examined the influence of genetic composition on body fat reduction induced by lifestyle changes using genetic risk scores (GRSs) of three phenotypes: GRS-carbohydrate (GRS-C), GRS-fat (GRS-F), and GRS-exercise (GRS-E). Our results showed that lifestyle modifications affected BFM more significantly in the high GRS class compared to the low GRS class, indicating the role of genetic factors affecting the efficiency of the lifestyle modification-induced BFM changes. Interestingly, the influence of exercise modification in the low GRS class with active lifestyle change was lower than that in the high GRS class with inactive lifestyle change (P = 0.022), suggesting the implication of genetic factors for efficient body fat control.

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3 low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, "exhausted" T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.

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 1000 unaffected controls (total n = 1,296) after data cleaning and filtering as well as imputation of non-genotyped 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.

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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 < 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 < 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.

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 1-2 weeks (1W) and 7-8 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 ≥ 5 μg/mL showed an increased species richness compared with patients with TLI < 5 μg/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.

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.

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&#95;Min&#95;hist), the histogram of the energy feature from outer ROIs (OUTER&#95;Energy&#95;Hist), the maximum probability of gray-level co-occurrence matrix (GLCM) feature from inner ROIs (INNER&#95;MaxProb&#95;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: 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.

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 1 , 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.

Intratumoral heterogeneity (ITH) refers to the presence of distinct tumor cell populations. It provides vital information for the clinical prognosis, drug responsiveness, and personalized treatment of cancer patients. As genomic ITH in various cancers affects the expression patterns of genes, the expression profile could be utilized for determining ITH level. Herein, we present a novel approach to directly detect high ITH defined as a larger number of subclones from the gene expression pattern through machine learning approaches. We examined associations between gene expression profile and ITH of 12 cancer types from The Cancer Genome Atlas (TCGA) database. Using stomach adenocarcinoma (STAD) showing high association, we evaluated the performance of our method in predicting ITH by employing three machine learning algorithms using gene expression profile data. We classified tumors into high and low heterogeneity groups using the learning model through the selection of LASSO feature. The result showed that support vector machines (SVMs) outperformed other algorithms (AUC = 0.84 in SVMs and 0.82 in Naïve Bayes) and we were able to improve predictive power by using both combined data from mutation and expression. Furthermore, we evaluated the prediction ability of each model using simulation data generated by mixing cell lines of the Cancer Cell Line Encyclopedia (CCLE), and obtained consistent results with using real dataset. Our approach could be utilized for discriminating tumors with heterogeneous cell populations to characterize ITH., Competing Interests: The authors declare that they have no competing interests.

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 < 0.001). We observed no specific correlation with non-synonymous mutations and no changes in the single-nucleotide variant spectrum after CCRT. Post-CCRT samples were enriched in gene sets related to immune signaling pathways, such as interferon gamma signaling and CD28 co-stimulation. Multiplex IHC showed an incremental trend in the proportion of CD4 positive cells in cytokeratin positive region after CCRT. However, CD8, CD20, FOXP1, PD-L1 showed no definitive trend. Proportion of immune cells calculated by CIBERSORT, showed that significant increase in neutrophils after CCRT., Conclusions: We have comprehensively analyzed the early immunogenomic changes induced in ESCC by CCRT and correlated them with clinical outcomes. Our results provide a potential basis for combining immunotherapy with CCRT for the treatment of ESCC.

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.

Purpose: This study investigated the correlations between parameters of 18 F-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., Methods: 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., Results: 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 SUV max 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 SUV peak 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., Conclusions: 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.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (<10%). Patients were classified into three categories based on the mutation patterns after cancer treatment. A significant association between the major mutation patterns and clinical outcome was observed. Patients whose relapsed tumor had fewer mutations than the diagnostic sample tended to be older, had longer progression-free survival, and achieved complete remission after relapse. Contrastingly, patients whose genetic profile only had concordant mutations without any change had the worst outcome., Conclusions: We characterized genomic changes in recurrent pediatric solid tumors. These findings could help to understand the biology of relapsed childhood cancer and to develop personalized treatment based on their genetic profile., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

[This corrects the article DOI: 10.18632/oncotarget.14928.].

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 < 0.001 for both NOTCH4 and TGFBR3). In addition, five novel fusion gene loci were identified in four lung adenocarcinomas. However, no significant virus-associated transcripts were detected in tumors. In conclusions, EGFR, IDH2, TP53, PTEN, EPHB4, and BRAF were identified as putative driver mutations of ground-glass nodular adenocarcinomas. Five novel fusion genes were also identified in four tumors. Viruses do not appear to be involved in the tumorigenesis of ground-glass nodular lung adenocarcinoma.

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level., (© 2018 Han et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: Tumors continuously evolve to maintain growth; secondary mutations facilitate this process, resulting in high tumor heterogeneity. In this study, we compared mutations in paired primary and metastatic colorectal cancer tumor samples to determine whether tumor heterogeneity can predict tumor metastasis. Experimental Design: Somatic variations in 46 pairs of matched primary-liver metastatic tumors and 42 primary tumors without metastasis were analyzed by whole-exome sequencing. Tumor clonality was estimated from single-nucleotide and copy-number variations. The correlation between clinical parameters of patients and clonal heterogeneity in liver metastasis was evaluated. Results: Tumor heterogeneity across colorectal cancer samples was highly variable; however, a high degree of tumor heterogeneity was associated with a worse disease-free survival. Highly heterogeneous primary colorectal cancer was correlated with a higher rate of liver metastasis. Recurrent somatic mutations in APC, TP53 , and KRAS were frequently detected in highly heterogeneous colorectal cancer. The variant allele frequency of these mutations was high, while somatic mutations in other genes such as PIK3CA and NOTCH1 were low. The number and distribution of primary colorectal cancer subclones were preserved in metastatic tumors. Conclusions: Heterogeneity of primary colorectal cancer tumors can predict the potential for liver metastasis and thus, clinical outcome of patients. Clin Cancer Res; 23(23); 7209-16. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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.

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.

Customized gene-panel tests, based on next-generation sequencing, have demonstrated their usefulness in a plethora of clinical settings. As with other clinical diagnostic techniques, gene-panel sequencing for clinical purposes requires precise quality control (QC) measures to ensure its reliability. Only detected variants are currently recorded in clinical reports; however, identifying whether a nondetected variant is a true or false negative is regarded essential in a clinical setting and, thus, a comprehensive QC measure is in demand. Conventional QC metrics, such as mean coverage and uniformity, are considered inadequate for such an evaluation. As such, a more specific measure focused on clinically important variants is herein proposed. In this study, we suggest a new scoring method for assessing the quality of clinical gene-panel sequencing data, specifically for the detection of a set of single-nucleotide variants. The performance of the method was analyzed using 2295 clinical samples (1012 formalin-fixed, paraffin-embedded and 1283 fresh-frozen tissues), and was shown to provide additional information that conventional methods do not show, such as mean depth and uniformity. Customized sequencing protocols, which include QC criteria, have been optimized by each genomic laboratory. The pass rate scoring method proposed in this study provides an appropriate QC response variable for the customized panel, which strengthens the reliability of calls on clinically relevant variants implicated in clinical reports., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Aberrant gene expression in the gut mucosa might contribute to the initiation and progression of Crohn's disease (CD). RNA sequencing (RNA-seq) provides precise measurements of expression levels of transcripts and their isoforms. The aim of this study was to use RNA-seq to investigate transcriptomic differences and identify significantly differentially expressed transcripts in inflamed and noninflamed intestinal mucosa of CD patients., Methods: RNA-seq was performed on 13 pairs of inflamed and noninflamed intestinal mucosa from 13 CD patients and on sex-matched normal mucosa of 13 healthy controls. Significantly differentially expressed transcripts were validated by immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay., Results: RNA-seq revealed genome-wide transcriptomic differences between normal mucosa, noninflamed, and inflamed CD mucosa. Among 950 differentially expressed genes, 19 were up- or downregulated (upregulation: ANGPT2, CHN1, CPXM1, CPZ, CXCL1, FCN3, GJC1, HSD11B1, LZTS1, MEOX1, MMP12, PLA1A, SERPINE1, SGIP1, and TRPC4; downregulation: FAM189A1, PDE6A, SLC38A4, and HMGCS2) with statistical significance (p < 0.01 and q < 0.05). Among them, CXCL1 exhibited the highest fold change between groups. Immunohistochemistry for CXCL1 revealed no expression in normal mucosa, slightly increased expression in noninflamed CD mucosa, and highly increased expression in inflamed CD mucosa. Quantitative reverse transcriptase polymerase chain reaction showed that CXCL1 expression was significantly associated with epithelial damage, increased infiltration of polymorphonuclear leukocytes, and submucosal fibrosis. Serum CXCL1 concentration measured by enzyme-linked immunosorbent assay was better correlated with CD activity index (r = 0.660) than with C-reactive protein (r = 0.204)., Conclusions: RNA-seq revealed transcriptomic differences between normal mucosa, noninflamed CD mucosa, and inflamed CD mucosa. Intestinal and serum CXCL1 was substantially increased with CD activity and can be used as a potential biomarker of CD.

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.

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.

Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients' blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

Tumor heterogeneity influences the clinical outcome of patients with cancer, and the diagnostic method to measure the tumor heterogeneity needs to be developed. We analyzed genomic features on pairs of primary and multiple metastatic lymph nodes from six patients with lung cancer using whole-exome sequencing and RNA sequencing. Although somatic single-nucleotide variants were shared in primary lung cancer and metastases, tumor evolution predicted by the pattern of genomic alterations was matched to anatomic location of the tumors. Four of six cases exhibited a branched clonal evolution pattern. Lymph nodes with acquired somatic variants demonstrated resistance to the cancer treatment. In this study, we demonstrated that multiple biopsies and sequencing strategies for different tumor regions are required for a comprehensive understanding of the landscape of genetic alteration and for guiding targeted therapy in advanced primary lung cancer. Cancer Res; 76(22); 6568-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

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.

Nephronophthisis-related ciliopathy (NPHP-RC) is a common genetic cause of end-stage renal failure during childhood and adolescence and exhibits an autosomal recessive pattern of inheritance. Genetic diagnosis is quite limited owing to genetic heterogeneity in NPHP-RC. We designed a novel approach involving the step-wise screening of Sanger sequencing and targeted exome sequencing for the genetic diagnosis of 55 patients with NPHP-RC. First, five NPHP-RC genes were analyzed by Sanger sequencing in phenotypically classified patients. Known pathogenic mutations were identified in 12 patients (21.8%); homozygous deletions of NPHP1 in 4 juvenile nephronophthisis patients, IQCB1/NPHP5 mutations in 3 Senior-Løken syndrome patients, a CEP290/NPHP6 mutation in 1 Joubert syndrome patient, and TMEM67/MKS3 mutations in 4 Joubert syndrome patients with liver involvement. In the remaining undiagnosed patients, we applied targeted exome sequencing of 34 ciliopathy-related genes to detect known pathogenic mutations in 7 (16.3%) of 43 patients. Another 18 likely damaging heterozygous variants were identified in 13 NPHP-RC genes in 18 patients. In this study, we report a variety of pathogenic and candidate mutations identified in 55 patients with NPHP-RC in Korea using a step-wise application of two genetic tests. These results support the clinical utility of targeted exome sequencing to resolve the issue of allelic and genetic heterogeneity in NPHP-RC.

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., Competing Interests: The authors declare that they have no competing interests.