Neutrophil diversity is associated with T-cell immunity and clinical relevance in patients with thyroid cancer.

Neutrophil heterogeneity is involved in autoimmune diseases, sepsis, and several cancers. However, the link between neutrophil heterogeneity and T-cell immunity in thyroid cancer is incompletely understood. We investigated the circulating neutrophil heterogeneity in 3 undifferentiated thyroid cancer (UTC), 14 differentiated thyroid cancer (DTC) (4 Stage IV, 10 Stage I-II), and healthy controls (n = 10) by transcriptomic data and cytometry. Participants with UTC had a significantly higher proportion of immature high-density neutrophils (HDN) and lower proportion of mature HDN in peripheral blood compared to DTC. The proportion of circulating PD-L1+ immature neutrophils were significantly increased in advanced cancer patients. Unsupervised analysis of transcriptomics data from circulating HDN revealed downregulation of innate immune response and T-cell receptor signaling pathway in cancer patients. Moreover, UTC patients revealed the upregulation of glycolytic process and glutamate receptor signaling pathway. Comparative analysis across tumor types and stages revealed the downregulation of various T-cell-related pathways, such as T-cell receptor signaling pathway and T-cell proliferation in advanced cancer patients. Moreover, the proportions of CD8+ and CD4+ T effector memory CD45RA+ (TEMRA) cells from peripheral blood were significantly decreased in UTC patients compared to DTC patients. Finally, we demonstrated that proportions of tumor-infiltrated neutrophils were increased and related with poor prognosis in advanced thyroid cancer using data from our RNA-seq and TCGA (The Cancer Genome Atlas) data. In conclusion, observed prevalence of circulating immature high-density neutrophils and their immunosuppressive features in undifferentiated thyroid cancers underscore the importance of understanding neutrophil dynamics in the context of tumor progression in thyroid cancer.

Transcriptomic characteristics according to tumor size and SUVmax in papillary thyroid cancer patients.

The SUVmax is a measure of FDG uptake and is related with tumor aggressiveness in thyroid cancer, however, its association with molecular pathways is unclear. Here, we investigated the relationship between SUVmax and gene expression profiles in 80 papillary thyroid cancer (PTC) patients. We conducted an analysis of DEGs and enriched pathways in relation to SUVmax and tumor size. SUVmax showed a positive correlation with tumor size and correlated with glucose metabolic process. The genes that indicate thyroid differentiation, such as SLC5A5 and TPO, were negatively correlated with SUVmax. Unsupervised analysis revealed that SUVmax positively correlated with DNA replication(r = 0.29, p = 0.009), pyrimidine metabolism(r = 0.50, p < 0.0001) and purine metabolism (r = 0.42, p = 0.0001). Based on subgroups analysis, we identified that PSG5, TFF3, SOX2, SL5A5, SLC5A7, HOXD10, FER1L6, and IFNA1 genes were found to be significantly associated with tumor aggressiveness. Both high SUVmax PTMC and macro-PTC are enriched in pathways of DNA replication and cell cycle, however, gene sets for purine metabolic pathways are enriched only in high SUVmax macro-PTC but not in high SUVmax PTMC. Our findings demonstrate the molecular characteristics of high SUVmax tumor and metabolism involved in tumor growth in differentiated thyroid cancer.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses.

The role of the serine/glycine metabolic pathway (SGP) has recently been demonstrated in tumors; however, the pathological relevance of the SGP in thyroid cancer remains unexplored. Here, we perform metabolomic profiling of 17 tumor-normal pairs; bulk transcriptomics of 263 normal thyroid, 348 papillary, and 21 undifferentiated thyroid cancer samples; and single-cell transcriptomes from 15 cases, showing the impact of mitochondrial one-carbon metabolism in thyroid tumors. High expression of serine hydroxymethyltransferase-2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is associated with low thyroid differentiation scores and poor clinical features. A subpopulation of tumor cells with high mitochondrial one-carbon pathway activity is observed in the single-cell dataset. SHMT2 inhibition significantly compromises mitochondrial respiration and decreases cell proliferation and tumor size in vitro and in vivo. Collectively, our results highlight the importance of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer and suggest that SHMT2 is a potent therapeutic target.

Adenosine Deaminase 2 Deficiency Caused by Biallele Variants Including Splicing Variant: The First Case in Korea.

Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by pathogenic variants of the ADA2 gene and has similar clinical features to polyarteritis nodosa (PAN). We, herein, report a case of DADA2 in Korea that was diagnosed in a patient with childhood-onset PAN. The patient had a truncal ataxia and facial palsy caused by thalamic infarction at 34 months of age. Livedo reticularis with Raynaud phenomenon and abdominal pain with fever were followed. Radiologic examination showed multiple infarctions in brain and kidney. She was diagnosed with PAN using skin biopsy and angiography. She had severe hemorrhagic strokes despite medical treatments. Her disease activity was controlled after adding a tumor necrosis factor-α inhibitor. Molecular analysis revealed compound heterozygous pathogenic variants of ADA2 gene. This is the first case of DADA2 in Korea. Genetic analysis for ADA2 gene should be considered in patients with childhood-onset PAN.

Severe protein C deficiency in a newborn caused by a homozygous pathogenic variant in the PROC gene: a case report.

BACKGROUND: Severe protein C deficiency is a rare and inherited cause of thrombophilia in neonates. Protein C acts as an anticoagulant, and its deficiency results in vascular thrombosis. Herein, we report a case of protein C deficiency with a homozygous pathogenic variant in a term neonate, with good outcomes after proper treatment. CASE PRESENTATION: A four-day-old male newborn was transferred to the Seoul National University Hospital on account of dark red to black skin lesions. He was born full-term with an average birth weight without perinatal problems. There were no abnormal findings in the prenatal tests, including intrauterine sonography. The first skin lesion was observed on his right toes and rapidly progressed to proximal areas, such as the lower legs, left arm, and buttock. Under the impression of thromboembolism or vasculitis, we performed a coagulopathy workup, which revealed a high D-dimer level of 23.05 µg/ml. A skin biopsy showed fibrin clots in most capillaries, and his protein C activity level was below 10%, from which we diagnosed protein C deficiency. On postnatal day 6, he experienced an apnea event with desaturation and an abnormal right pupillary light reflex. Brain computed tomography showed multifocal patchy intracranial hemorrhage and intraventricular hemorrhage with an old ischemic lesion. Ophthalmic examination revealed bilateral retinal traction detachments with retinal folds. Protein C concentrate replacement therapy was added to previous treatments including steroids, prostaglandin E1, and anticoagulation. After replacement therapy, there were no new skin lesions, and the previous lesions recovered with scarring. Although there were no new brain hemorrhagic infarctions, there was ongoing ischemic tissue loss, which required further rehabilitation. Ophthalmic surgical interventions were performed to treat the bilateral retinal traction detachments with retinal folds. Molecular analysis revealed a homozygous pathogenic variant in the PROC gene. CONCLUSION: Severe protein C deficiency can manifest as a fatal coagulopathy in any organ. Early diagnosis and proper treatment, including protein C concentrate replacement, may improve outcomes without serious sequelae.

Acquired Resistance to Third-Generation EGFR Tyrosine Kinase Inhibitors in Patients With De Novo EGFRT790M-Mutant NSCLC.

INTRODUCTION: EGFRT790M mostly exists subclonally and is acquired as the most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKIs). Nevertheless, because de novo EGFRT790M-mutant NSCLC is rare, little is known on acquired resistance mechanisms to third-generation EGFR TKIs. METHODS: Acquired resistance mechanisms were analyzed using tumor and plasma samples before and after third-generation EGFR TKI treatment in four patients with de novo EGFRT790M-mutant NSCLC. Genetic alterations were analyzed by whole-exome sequencing, targeted sequencing, fluorescence in situ hybridization, and droplet digital PCR. MTORL1433S, confirmed for oncogenicity using the Ba/F3 system, was reproduced in H1975 cell lines using CRISPR/Cas9-RNP. RESULTS: Of seven patients with NSCLC with de novo EGFRT790M/L858R mutation, four (LC1-4) who received third-generation EGFR TKIs acquired resistance after achieving a partial response (median = 27 mo, range: 17-48 mo). Novel MTORL1433S and EGFRC797S/L798I mutations in cis, MET amplification, and EGFRC797S mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs. The MTORL1433S mutation was oncogenic in Ba/F3 models and revealed resistance to osimertinib through AKT signaling activation in NCI-H1975 cells harboring the MTORL1433S mutation edited by CRISPR/Cas9 (half-maximal inhibitory concentration, 800 ± 67 nM). Osimertinib in combination with mTOR inhibitors abrogated acquired resistance to osimertinib. CONCLUSIONS: Activation of bypass pathways and the EGFRC797S or EGFRC797S/L798I mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs in patients with NSCLC with de novo EGFRT790M mutation. In addition, MTORL1433S- and EGFRL858R/T790M-mutant NSCLC cells were sensitive to osimertinib plus mTOR inhibitors.

Implication of CD69+ CD103+ tissue-resident-like CD8+ T cells as a potential immunotherapeutic target for cholangiocarcinoma.

BACKGROUND: The heterogeneous immune landscapes of intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Here we aimed to investigate the implications of tissue-resident memory (TRM)-related features of tumour-infiltrating CD8+ T cells (CD8+ TILs) from ICC patients. METHODS: From ICC patients, we obtained blood samples and ICC surgical specimens (n = 33). We performed multicolour flow cytometry, multiplexed immunohistochemistry and RNA sequencing. RESULTS: When compared to peripheral CD8+ T cells, the CD8+ TILs included significantly higher proportions of the CD69+ CD103- and CD69+ CD103+ TRM-like subsets (P < .001 for both). Relative to CD69- and CD69+ CD103- cells, the CD69+ CD103+ CD8+ TILs harboured higher levels of T-cell markers representing tumour specificity (ie CD39), proliferation (ie Ki-67) and T-cell activation (ie HLA-DR and CD38) (all P < .001). Moreover, compared to the stroma, the tumour margin and core density each had a significantly higher density of CD103+ CD8+ TILs (P < .001 for both). ICCs with high proportions of CD69+ CD103+ cells displayed higher levels of parameters associated with response to immune checkpoint inhibitors (ICIs)-including number of CD8+ TIL infiltrates (P = .019), PD-L1 expression in the tumour (P = .046) and expression of the T cell-inflamed gene signature (P < .001). ICCs with lower proportions of CD69+ CD103+ CD8+ TILs exhibited significant enrichment of genes related to the Wnt/ß-catenin (P < .001) and TGF-ß pathways (P = .002). CONCLUSION: CD69+ CD103+ TRM-like CD8+ TILs represent prominent tumour-specific immune responses and hold promise as a potential therapeutic target in ICC patients. Differential TRM-related features of ICCs may help develop future immunotherapeutic strategies such as maximizing TRM responses or inhibiting pathways contributing to immune evasion.

4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39+ CD8 T cells from primary and metastatic sites of epithelial ovarian cancers.

BACKGROUND: Responses to immunotherapy vary between different cancer types and sites. Here, we aimed to investigate features of exhaustion and activation in tumor-infiltrating CD8 T cells at both the primary and metastatic sites in epithelial ovarian cancer. METHODS: Tumor tissues and peripheral blood were obtained from 65 patients with ovarian cancer. From these samples, we isolated tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells. These cells were used for immunophenotype using multicolor flow cytometry, gene expression profile using RNA sequencing and ex vivo functional restoration assays. RESULTS: We found that CD39+ CD8 TILs were enriched with tumor-specific CD8 TILs, and that the activation status of these cells was determined by the differential programmed cell death protein 1 (PD-1) expression level. CD39+ CD8 TILs with high PD-1 expression (PD-1high) exhibited features of highly tumor-reactive and terminally exhausted phenotypes. Notably, PD-1high CD39+ CD8 TILs showed similar characteristics in terms of T-cell exhaustion and activation between the primary and metastatic sites. Among co-stimulatory receptors, 4-1BB was exclusively overexpressed in CD39+ CD8 TILs, especially on PD-1high cells, and 4-1BB-expressing cells displayed immunophenotypes indicating higher degrees of T-cell activation and proliferation, and less exhaustion, compared with cells not expressing 4-1BB. Importantly, 4-1BB agonistic antibodies further enhanced the anti-PD-1-mediated reinvigoration of exhausted CD8 TILs from both primary and metastatic sites. CONCLUSION: Severely exhausted PD-1high CD39+ CD8 TILs displayed a distinctly heterogeneous exhaustion and activation status determined by differential 4-1BB expression levels, providing rationale and evidence for immunotherapies targeting co-stimulatory receptor 4-1BB in ovarian cancers.

The genome-wide landscape of C:G > T:A polymorphism at the CpG contexts in the human population.

BACKGROUND: The C:G > T:A substitution at the CpG dinucleotide contexts is the most frequent substitution type in genome evolution. The mutational process is obviously ongoing in the human germline; however, its impact on common and rare genomic polymorphisms has not been comprehensively investigated yet. Here we observed the landscape and dynamics of C:G > T:A substitutions from population-scale human genome sequencing datasets including ~ 4300 whole-genomes from the 1000 Genomes and the pan-cancer analysis of whole genomes (PCAWG) Project and ~ 60,000 whole-exomes from the Exome Aggregation Consortium (ExAC) database. RESULTS: Of the 28,084,558 CpG sites in the human reference genome, 26.0% show C:G > T:A substitution in the dataset. Remarkably, CpGs in CpG islands (CGIs) have a much lower frequency of such mutations (5.6%). Interestingly, the mutation frequency of CGIs is not uniform with a significantly higher C:G > T:A substitution rate for intragenic CGIs compared to other types. For non-CGI CpGs, the mutation rate was positively correlated with the distance from the nearest CGI up to 2 kb. Finally, we found the impact of negative selection for coding CpG mutations resulting in amino acid change. CONCLUSIONS: This study provides the first unbiased rate of C:G > T:A substitution at the CpG dinucleotide contexts, using population-scale human genome sequencing data. Our findings provide insights into the dynamics of the mutation acquisition in the human genome.

Dll4 Suppresses Transcytosis for Arterial Blood-Retinal Barrier Homeostasis.

RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

4-1BB Delineates Distinct Activation Status of Exhausted Tumor-Infiltrating CD8+ T Cells in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Targeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4-1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor-infiltrating CD8+ T cells (CD8+ tumor-infiltrating lymphocytes [TILs]) and its association with distinct T-cell activation features among exhausted CD8+ TILs in hepatocellular carcinoma (HCC). APPROACH AND RESULTS: Tumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA-sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4-1BB was most prominently expressed on CD8+ TILs. 4-1BB expression was almost exclusively detected on CD8+ T cells in the tumor-especially on programmed death 1 (PD-1)high cells and not PD-1int and PD-1neg cells. Compared to PD-1int and 4-1BBneg PD-1high CD8+ TILs, 4-1BBpos PD-1high CD8+ TILs exhibited higher levels of tumor reactivity and T-cell activation markers and significant enrichment for T-cell activation gene signatures. Per-patient analysis revealed positive correlations between percentages of 4-1BBpos cells among CD8+ TILs and levels of parameters of tumor reactivity and T-cell activation. Among highly exhausted PD-1high CD8+ TILs, 4-1BBpos cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4-1BB-related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4-1BB agonistic antibodies enhanced the function of CD8+ TILs and further enhanced the anti-PD-1-mediated reinvigoration of CD8+ TILs, especially in cases showing high levels of T-cell activation. CONCLUSION: 4-1BB expression on CD8+ TILs represents a distinct activation state among highly exhausted CD8+ T cells in HCC. 4-1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T-cell activation.

MK5 Regulates YAP Stability and Is a Molecular Target in YAP-Driven Cancers.

Transcriptional regulator YAP is activated in multiple human cancers and plays critical roles in tumor initiation, progression, metastasis, and drug resistance. However, therapeutic targeting of the Hippo-YAP pathway has been challenging due to its low druggability and limited knowledge of YAP regulation in cancer. Here we present a functional screen and identify a novel therapeutic target for YAP-driven tumorigenesis. RNAi screening using an oncogenic YAP activation model identified the serine/threonine kinase MK5 as a positive regulator of YAP. MK5 physically interacted with YAP and counteracted CK1δ/ε-mediated YAP ubiquitination and degradation independent of LATS1/2. MK5 kinase activity was essential for protecting YAP from ubiquitin-mediated degradation and cytoplasmic retention. Downregulating MK5 expression inhibited the survival of YAP-activated cancer cell lines and mouse xenograft models. MK5 upregulation was associated with high levels of YAP expression and poor prognosis in clinical tumor samples, confirming its important role for YAP activity in human cancer. These results uncover MK5 as a novel factor that regulates YAP stability, and targeting the YAP degradation pathway controlled by MK5 is a potential strategy for suppressing YAP activity in cancer. SIGNIFICANCE: These findings reveal MK5 is a novel kinase that regulates YAP in a LATS-independent manner and can be targeted for cancer therapy.

Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma.

Mutational processes giving rise to lung adenocarcinomas (LADCs) in non-smokers remain elusive. We analyzed 138 LADC whole genomes, including 83 cases with minimal contribution of smoking-associated mutational signature. Genomic rearrangements were not correlated with smoking-associated mutations and frequently served as driver events of smoking-signature-low LADCs. Complex genomic rearrangements, including chromothripsis and chromoplexy, generated 74% of known fusion oncogenes, including EML4-ALK, CD74-ROS1, and KIF5B-RET. Unlike other collateral rearrangements, these fusion-oncogene-associated rearrangements were frequently copy-number-balanced, representing a genomic signature of early oncogenesis. Analysis of mutation timing revealed that fusions and point mutations of canonical oncogenes were often acquired in the early decades of life. During a long latency, cancer-related genes were disrupted or amplified by complex rearrangements. The genomic landscape was different between subgroups-EGFR-mutant LADCs had frequent whole-genome duplications with p53 mutations, whereas fusion-oncogene-driven LADCs had frequent SETD2 mutations. Our study highlights LADC oncogenesis driven by endogenous mutational processes.

Association Between Expression Level of PD1 by Tumor-Infiltrating CD8+ T Cells and Features of Hepatocellular Carcinoma.

BACKGROUND & AIMS: T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8+ T cells isolated from HCC specimens. METHODS: We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8+ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8+ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8+ T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry. RESULTS: PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1+, TBEThigh/eomesoderminlow, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8+ T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8+ T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. CONCLUSIONS: We found HCC specimens to contain CD8+ T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8+ T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8+ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

Mutalisk: a web-based somatic MUTation AnaLyIS toolKit for genomic, transcriptional and epigenomic signatures.

Somatic genome mutations occur due to combinations of various intrinsic/extrinsic mutational processes and DNA repair mechanisms. Different molecular processes frequently generate different signatures of somatic mutations in their own favored contexts. As a result, the regional somatic mutation rate is dependent on the local DNA sequence, the DNA replication/RNA transcription dynamics and epigenomic chromatin organization landscape in the genome. Here, we propose an online computational framework, termed Mutalisk, which correlates somatic mutations with various genomic, transcriptional and epigenomic features in order to understand mutational processes that contribute to the generation of the mutations. This user-friendly tool explores the presence of localized hypermutations (kataegis), dissects the spectrum of mutations into the maximum likelihood combination of known mutational signatures and associates the mutation density with numerous regulatory elements in the genome. As a result, global patterns of somatic mutations in any query sample can be efficiently screened, thus enabling a deeper understanding of various mutagenic factors. This tool will facilitate more effective downstream analyses of cancer genome sequences to elucidate the diversity of mutational processes underlying the development and clonal evolution of cancer cells. Mutalisk is freely available at http://mutalisk.org.

Sox7 promotes high-grade glioma by increasing VEGFR2-mediated vascular abnormality.

High-grade glioma (HGG) is highly angiogenic, but antiangiogenic therapy has transient clinical benefit in only a fraction of patients. Vascular regulators of these heterogeneous responses remain undetermined. We found up-regulation of Sox7 and down-regulation of Sox17 in tumor endothelial cells (tECs) in mouse HGG. Sox7 deletion suppressed VEGFR2 expression, vascular abnormality, hypoxia-driven invasion, regulatory T cell infiltration, and tumor growth. Conversely, Sox17 deletion exacerbated these phenotypes by up-regulating Sox7 in tECs. Anti-VEGFR2 antibody treatment delayed tumor growth by normalizing Sox17-deficient abnormal vessels with high Sox7 levels but promoted it by regressing Sox7-deficient vessels, recapitulating variable therapeutic responses to antiangiogenic therapy in HGG patients. Our findings establish that Sox7 promotes tumor growth via vessel abnormalization, and its level determines the therapeutic outcome of VEGFR2 inhibition in HGG. In 189 HGG patients, Sox7 expression was heterogeneous in tumor vessels, and high Sox7 levels correlated with poor survival, early recurrence, and impaired vascular function, emphasizing the clinical relevance of Sox7 in HGG.

Alterations in the Rho pathway contribute to Epstein-Barr virus-induced lymphomagenesis in immunosuppressed environments.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas (EBV+-DLBLs) tend to occur in immunocompromised patients, such as the elderly or those undergoing solid organ transplantation. The pathogenesis and genomic characteristics of EBV+-DLBLs are largely unknown because of the limited availability of human samples and lack of experimental animal models. We observed the development of 25 human EBV+-DLBLs during the engraftment of gastric adenocarcinomas into immunodeficient mice. An integrated genomic analysis of the human-derived EBV+-DLBLs revealed enrichment of mutations in Rho pathway genes, including RHPN2, and Rho pathway transcriptomic activation. Targeting the Rho pathway using a Rho-associated protein kinase (ROCK) inhibitor, fasudil, markedly decreased tumor growth in EBV+-DLBL patient-derived xenograft (PDX) models. Thus, alterations in the Rho pathway appear to contribute to EBV-induced lymphomagenesis in immunosuppressed environments.

Prospective Evaluation of Changes in Tumor Size and Tumor Metabolism in Patients with Advanced Gastric Cancer Undergoing Chemotherapy: Association and Clinical Implication.

A change in tumor size is a well-validated and commonly used value for evaluating response to chemotherapy in cancer. Metabolic changes induced by chemotherapy are related to prognosis in several tumor types. However, the clinical implication of metabolic changes in patients with advanced gastric cancer (AGC) undergoing chemotherapy remains unclear. We aimed to evaluate response of tumor size and metabolism in AGC during chemotherapy and to reveal the relationship between them in view of their impact on patient survival. Methods: We prospectively enrolled patients with AGC before the initiation of first-line palliative chemotherapy. Using baseline and follow-up contrast-enhanced CT and 18F-FDG PET, we assessed the tumor diameter, SUVmax, and total lesion glycolysis in each lesion and their changes during chemotherapy at the same time. We included all lesions with the maximal longest diameters over 1 cm on CT, and each lesion was evaluated by matched 18F-FDG PET. We analyzed the association between changes in tumor metabolism and tumor size and performed outcome analysis on overall survival (OS) and progression-free survival (PFS). Results: Seventy-four patients were enrolled, and the number of all lesions included in this study was 620. Compared with adenocarcinomas, poorly cohesive carcinomas demonstrated lower SUVmax irrespective of tumor size (P < 0.001). Human epidermal growth factor receptor 2 (HER2)-positive tumors showed higher SUVmax than HER2-negative tumors (P = 0.002). The changes in SUVmax due to chemotherapy had a linear correlation with the changes in tumor size of each lesion, and a 30% tumor size reduction was associated with a 50% SUVmax reduction (P < 0.001). Total lesion glycolysis changes also correlated with tumor size changes (P < 0.001). Better OS and PFS were obtained in patients with both tumor size and SUVmax reduction than in patients with either size or SUVmax reduction only (OS, P = 0.003; PFS, P = 0.038). Conclusion: Changes in tumor metabolism induced by chemotherapy correlated with changes in tumor size in AGC. Considering both changes in metabolism and size could help predict a more accurate prognosis for AGC patients undergoing chemotherapy.

Clonal History and Genetic Predictors of Transformation Into Small-Cell Carcinomas From Lung Adenocarcinomas.

Purpose Histologic transformation of EGFR mutant lung adenocarcinoma (LADC) into small-cell lung cancer (SCLC) has been described as one of the major resistant mechanisms for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the molecular pathogenesis is still unclear. Methods We investigated 21 patients with advanced EGFR-mutant LADCs that were transformed into EGFR TKI-resistant SCLCs. Among them, whole genome sequencing was applied for nine tumors acquired at various time points from four patients to reconstruct their clonal evolutionary history and to detect genetic predictors for small-cell transformation. The findings were validated by immunohistochemistry in 210 lung cancer tissues. Results We identified that EGFR TKI-resistant LADCs and SCLCs share a common clonal origin and undergo branched evolutionary trajectories. The clonal divergence of SCLC ancestors from the LADC cells occurred before the first EGFR TKI treatments, and the complete inactivation of both RB1 and TP53 were observed from the early LADC stages in sequenced tumors. We extended the findings by immunohistochemistry in the early-stage LADC tissues of 75 patients treated with EGFR TKIs; inactivation of both Rb and p53 was strikingly more frequent in the small-cell-transformed group than in the nontransformed group (82% v 3%; odds ratio, 131; 95% CI, 19.9 to 859). Among patients registered in a predefined cohort (n = 65), an EGFR mutant LADC that harbored completely inactivated Rb and p53 had a 43× greater risk of small-cell transformation (relative risk, 42.8; 95% CI, 5.88 to 311). Branch-specific mutational signature analysis revealed that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-induced hypermutation was frequent in the branches toward small-cell transformation. Conclusion EGFR TKI-resistant SCLCs are branched out early from the LADC clones that harbor completely inactivated RB1 and TP53. The evaluation of RB1 and TP53 status in EGFR TKI-treated LADCs is informative in predicting small-cell transformation.