Super enhancer loci of EGFR regulate EGFR variant 8 through enhancer RNA and strongly associate with survival in HNSCCs.

Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in human cancers due to mutation, amplification, and epigenetic hyperactivity, which leads to deregulated transcriptional mechanism. Among the eight different EGFR isoforms, the mechanism of regulation of full-length variant 1 is well-known, no studies have examined the function & factors regulating the expression of variant 8. This study aimed to understand the function of EGFR super-enhancer loci and its associated transcription factors regulating the expression of EGFR variant 8. Our study shows that overexpression of variant 8 and its transcription was more prevalent than variant 1 in many cancers and positively correlated with the EGFR-AS1 expression in oral cancer and HNSCC. Notably, individuals overexpressing variant 8 showed shorter overall survival and had a greater connection with other clinical traits than patients with overexpression of variant 1. In this study, TCGA enhancer RNA profiling on the constituent enhancer (CE1 and CE2) region revealed that the multiple enhancer RNAs formed from CE2 by employing CE1 as a promoter. Our bioinformatic analysis further supports the enrichment of enhancer RNA specific chromatin marks H3K27ac, H3K4me1, POL2 and H2AZ on CE2. GeneHancer and 3D chromatin capture analysis showed clustered interactions between CE1, CE2 loci and this interaction may regulates expression of both EGFR-eRNA and variant 8. Moreover, increased expression of SNAI2 and its close relationship to EGFR-AS1 and variant 8 suggest that SNAI2 could regulates variant 8 overexpression by building a MegaTrans complex with both EGFR-eRNA and EGFR-AS1. Our findings show that EGFR variant 8 and its transcriptional regulation & chromatin modification by eRNAs may provide a rationale for targeting RNA splicing in combination with targeted EGFR therapies in cancer.

Metagenomic analyses of 7000 to 5500 years old coprolites excavated from the Torihama shell-mound site in the Japanese archipelago.

Coprolites contain various kinds of ancient DNAs derived from gut micro-organisms, viruses, and foods, which can help to determine the gut environment of ancient peoples. Their genomic information should be helpful in elucidating the interaction between hosts and microbes for thousands of years, as well as characterizing the dietary behaviors of ancient people. We performed shotgun metagenomic sequencing on four coprolites excavated from the Torihama shell-mound site in the Japanese archipelago. The coprolites were found in the layers of the Early Jomon period, corresponding stratigraphically to 7000 to 5500 years ago. After shotgun sequencing, we found that a significant number of reads showed homology with known gut microbe, viruses, and food genomes typically found in the feces of modern humans. We detected reads derived from several types of phages and their host bacteria simultaneously, suggesting the coexistence of viruses and their hosts. The food genomes provide biological evidence for the dietary behavior of the Jomon people, consistent with previous archaeological findings. These results indicate that ancient genomic analysis of coprolites is useful for understanding the gut environment and lifestyle of ancient peoples.

Noninvasive graft monitoring using donor-derived cell-free DNA in Japanese liver transplantation.

AIM: To evaluate the use of donor-derived cell-free DNA (dd-cfDNA) in diagnosing graft injuries in Japanese liver transplantation (LTx), including family-related living donors. METHODS: A total of 321 samples from 10 newly operated LTx recipients were collected to monitor the early dynamics of dd-cfDNA levels after LTx. Fifty-five samples from 55 recipients were collected during protocol biopsies (PB), whereas 36 samples from 27 recipients were collected during event biopsies, consisting of 11 biopsy-proven acute rejection (AR), 20 acute dysfunctions without rejection (ADWR), and 5 chronic rejections. The levels of dd-cfDNA were quantified using a next-generation sequencer based on single nucleotide polymorphisms. RESULTS: The dd-cfDNA levels were elevated significantly after LTx, followed by a rapid decline to the baseline in patients without graft injury within 30 days post-LTx. The dd-cfDNA levels were significantly higher in the 11 samples obtained during AR than those obtained during PB (p < 0.0001), which decreased promptly after treatment. The receiver operator characteristic curve analysis of diagnostic ability yielded areas under the curve of 0.975 and 0.897 for AR (rejection activity index [RAI] ≥3) versus PB and versus non-AR (ADWR + PB). The dd-cfDNA levels during AR were elevated earlier and correlated more strongly with the RAI (r = 0.740) than aspartate aminotransferase/alanine aminotransferase. The dd-cfDNA levels were neither associated with graft fibrosis based on histology nor the status of donor-specific antibodies in PB samples. CONCLUSIONS: Donor-derived cell-free DNA serves as a sensitive biomarker for detecting graft injuries in LTx. Further large-scale cohort studies are warranted to optimize its use in differentiating various post-LTx etiologies.

Detection of APP gene recombinant in human blood plasma.

The pathogenesis of Alzheimer's disease (AD) is believed to involve the accumulation of amyloid-ß in the brain, which is produced by the sequential cleavage of amyloid precursor protein (APP) by ß-secretase and γ-secretase. Recently, analysis of genomic DNA and mRNA from postmortem brain neurons has revealed intra-exonic recombinants of APP (gencDNA), which have been implicated in the accumulation of amyloid-ß. In this study, we computationally analyzed publicly available sequence data (SRA) using probe sequences we constructed to screen APP gencDNAs. APP gencDNAs were detected in SRAs constructed from both genomic DNA and RNA obtained from the postmortem brain and in the SRA constructed from plasma cell-free mRNA (cf-mRNA). The SRA constructed from plasma cf-mRNA showed a significant difference in the number of APP gencDNA reads between SAD and NCI: the p-value from the Mann-Whitney U test was 5.14 × 10-6. The transcripts were also found in circulating nucleic acids (CNA) from our plasma samples with NGS analysis. These data indicate that transcripts of APP gencDNA can be detected in blood plasma and suggest the possibility of using them as blood biomarkers for Alzheimer's disease.

Angiogenic and inflammatory responses in human induced microglia-like (iMG) cells from patients with Moyamoya disease.

Angiogenic factors associated with Moyamoya disease (MMD) are overexpressed in M2 polarized microglia in ischemic stroke, suggesting that microglia may be involved in the pathophysiology of MMD; however, existing approaches are not applicable to explore this hypothesis. Herein we applied blood induced microglial-like (iMG) cells. We recruited 25 adult patients with MMD and 24 healthy volunteers. Patients with MMD were subdivided into progressive (N = 7) or stable (N = 18) group whether novel symptoms or radiographic advancement of Suzuki stage within 1 year was observed or not. We produced 3 types of iMG cells; resting, M1-, and M2-induced cells from monocytes, then RNA sequencing followed by GO and KEGG pathway enrichment analysis and qPCR assay were performed. RNA sequencing of M2-induced iMG cells revealed that 600 genes were significantly upregulated (338) or downregulated (262) in patients with MMD. Inflammation and immune-related factors and angiogenesis-related factors were specifically associated with MMD in GO analysis. qPCR for MMP9, VEGFA, and TGFB1 expression validated these findings. This study is the first to demonstrate that M2 microglia may be involved in the angiogenic process of MMD. The iMG technique provides a promising approach to explore the bioactivity of microglia in cerebrovascular diseases.

Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma.

Although the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT-SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT-SCCs, we performed whole-exome sequencing by collecting 16 samples from histologically different parts of two MCT-SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT-SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC-mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT-SCC.

AMBRA1 p.Gln30Arg Mutation, Identified in a Cowden Syndrome Family, Exhibits Hyperproliferative Potential in hTERT-RPE1 Cells.

Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.

Extraction of CRISPR-targeted sequences from the metagenome.

Homology-based search is commonly used to uncover mobile genetic elements (MGEs) from metagenomes, but it heavily relies on reference genomes in the database. Here we introduce a protocol to extract CRISPR-targeted sequences from the assembled human gut metagenomic sequences without using a reference database. We describe the assembling of metagenome contigs, the extraction of CRISPR direct repeats and spacers, the discovery of protospacers, and the extraction of protospacer-enriched regions using the graph-based approach. This protocol could extract numerous characterized/uncharacterized MGEs. For complete details on the use and execution of this protocol, please refer to Sugimoto et al. (2021).

The landscape of genetic alterations of UVB-induced skin tumors in DNA repair-deficient mice.

Non-melanoma skin cancer (NMSC) is mainly caused by ultraviolet (UV)-induced somatic mutations and is characterized by UV signature modifications. Xeroderma pigmentosum group A (Xpa) knockout mice exhibit extreme UV-induced photo-skin carcinogenesis, along with a photosensitive phenotype. We performed whole-exome sequencing (WES) of squamous cell carcinoma (SCC) samples after repetitive ultraviolet B (UVB) exposure to investigate the differences in the landscape of somatic mutations between Xpa knockout and wild-type mice. Although the tumors that developed in mice harboured UV signature mutations in a similar set of cancer-related genes, the pattern of transcriptional strand asymmetry was largely different; UV signature mutations in Xpa knockout and wild-type mice preferentially occurred in transcribed and non-transcribed strands, respectively, reflecting a deficiency in transcription-coupled nucleotide excision repair in Xpa knockout mice. Serial time point analyses of WES for a tumor induced by only a single UVB exposure showed pathogenic mutations in Kras, Fat1, and Kmt2c, which may be driver genes for the initiation and promotion of SCC in Xpa knockout mice. Furthermore, the inhibitory effects on tumor production in Xpa knockout mice by the anti-inflammatory CXCL1 monoclonal antibody affected the pattern of somatic mutations, wherein the transcriptional strand asymmetry was attenuated and the activated signal transduction was shifted from the RAS/RAF/MAPK to the PIK3CA pathway.

Detection of Ancient Viruses and Long-Term Viral Evolution.

The COVID-19 outbreak has reminded us of the importance of viral evolutionary studies as regards comprehending complex viral evolution and preventing future pandemics. A unique approach to understanding viral evolution is the use of ancient viral genomes. Ancient viruses are detectable in various archaeological remains, including ancient people's skeletons and mummified tissues. Those specimens have preserved ancient viral DNA and RNA, which have been vigorously analyzed in the last few decades thanks to the development of sequencing technologies. Reconstructed ancient pathogenic viral genomes have been utilized to estimate the past pandemics of pathogenic viruses within the ancient human population and long-term evolutionary events. Recent studies revealed the existence of non-pathogenic viral genomes in ancient people's bodies. These ancient non-pathogenic viruses might be informative for inferring their relationships with ancient people's diets and lifestyles. Here, we reviewed the past and ongoing studies on ancient pathogenic and non-pathogenic viruses and the usage of ancient viral genomes to understand their long-term viral evolution.

High incidence of PI3K pathway gene mutations in South Indian cervical cancers.

INTRODUCTION: Cervical cancer is the second most common cancer in India. The phosphatidylinositol-3 kinase (PI3K) signaling is one of the most commonly activated pathways in cancer and comprises key molecules commonly targeted in cancer therapy. This study analyzed six PI3K pathway gene mutations. METHODS: We carried out targeted next-generation sequencing of six PI3K pathway genes (PIK3CA, PIK3R1, PTEN, AKT1, TSC2, and mTOR) in a total of 93 South Indian cervical cancer samples and confirmed them by sanger sequencing. RESULTS: The PI3K pathway gene mutations were observed in 54.8% (51/93) of the tumors and PIK3CA was the most mutated (34.4%, 32/93), followed by TSC2 (18.3%, 17/93), and PIK3R1 (14%, 13/93). The PIK3CA hotspot mutations E542K and E545K observed in this study were likely to disrupt the p110α-p85α interaction that could result in the PI3K pathway activation. We also found a few novel mutations in PIK3R1, PTEN, AKT1, TSC2, and mTOR genes while some of the tumors harbored multiple mutations in the genes of the PI3K pathway. The majority of the tumors were positive for high-risk HPV16/18 (60.7%). CONCLUSIONS: The high incidence of the PI3K pathway gene mutations observed in this study could be exploited for the therapeutic management of cervical cancers.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium.

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that "rhizome structures", in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

Genome-wide meta-analysis between renal overload type and renal underexcretion type of clinically defined gout in Japanese populations.

Despite progress in understanding of the genetic basis of gout, the precise factors affecting differences in gout susceptibility among different gout subtypes remain unclear. Using clinically diagnosed gout patients, we conducted a genome-wide meta-analysis of two distinct gout subtypes: the renal overload type and the renal underexcretion type. We provide genetic evidence at a genome-wide level of significance that supports a positive association between ABCG2 dysfunction and acquisition of the renal overload type.

APOBEC mediated mutagenesis drives genomic heterogeneity in endometriosis.

Endometriosis is a benign gynecologic condition, acting as a precursor of certain histological subtypes of ovarian cancers. The epithelial cells of endometriotic tissues and normal uterine endometrium accumulated somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and Kirsten rat sarcoma (KRAS) proto-oncogene. To determine the genomic characteristic of endometriotic epithelial cells and normal uterine endometrium and to identify the predominant mutational process acting on them, we studied the somatic mutation profiles obtained from whole exome sequencing of 14 endometriotic epithelium and 11 normal uterine endometrium tissues and classified them into mutational signatures. We observed that single base substitutions 2/13 (SBS), attributed to Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit (APOBEC) induced mutagenesis, were significant in endometriotic tissues, but not in the normal uterine endometrium. Additionally, the larger number and wider allele frequency distribution of APOBEC signature mutations, compared to cancer-associated driver mutations in endometriotic epithelium suggested APOBEC mutagenesis as an important source of mutational burden and heterogeneity in endometriosis. Further, the relative risk of enriched APOBEC signature mutations was higher in endometriosis patients who were carriers of APOBEC3A/3B germline deletion, a common polymorphism in East Asians which involves the complete loss of APOBEC3B coding region. Our results illustrate the significance of APOBEC induced mutagenesis in driving the genomic heterogeneity of endometriosis.

Decoding the diversity of killer immunoglobulin-like receptors by deep sequencing and a high-resolution imputation method.

The killer cell immunoglobulin-like receptor (KIR) recognizes human leukocyte antigen (HLA) class I molecules and modulates the function of natural killer cells. Despite its role in immunity, the complex genomic structure has limited a deep understanding of the KIR genomic landscape. Here we conduct deep sequencing of 16 KIR genes in 1,173 individuals. We devise a bioinformatics pipeline incorporating copy number estimation and insertion or deletion (indel) calling for high-resolution KIR genotyping. We define 118 alleles in 13 genes and demonstrate a linkage disequilibrium structure within and across KIR centromeric and telomeric regions. We construct a KIR imputation reference panel (nreference = 689, imputation accuracy = 99.7%), apply it to biobank genotype (ntotal = 169,907), and perform phenome-wide association studies of 85 traits. We observe a dearth of genome-wide significant associations, even in immune traits implicated previously to be associated with KIR (the smallest p = 1.5 × 10-4). Our pipeline presents a broadly applicable framework to evaluate innate immunity in large-scale datasets.

Allelic and haplotypic HLA diversity in indigenous Malaysian populations explored using Next Generation Sequencing.

The heterogenous population of Malaysia includes more than 50 indigenous groups, and characterizing their HLA diversity would not only provide insights to their ancestry, but also on the effects of natural selection on their genome. We utilized hybridization-based sequence capture and short-read sequencing on the HLA region of 172 individuals representing seven indigenous groups in Malaysia (Jehai, Kintaq, Temiar, Mah Meri, Seletar, Temuan, Bidayuh). Allele and haplotype frequencies of HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1, and -DPB1 revealed several ancestry-informative markers. Using SNP-based heterozygosity and pairwise Fst, we observed signals of natural selection, particularly in HLA-A, -C and -DPB1 genes. Consequently, we showed the impact of natural selection on phylogenetic inference using HLA and non-HLA SNPs. We demonstrate the utility of Next Generation Sequencing for generating unambiguous, high-throughput, high-resolution HLA data that adds to our knowledge of HLA diversity and natural selection in indigenous minority groups.

Comprehensive discovery of CRISPR-targeted terminally redundant sequences in the human gut metagenome: Viruses, plasmids, and more.

Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse, and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes that characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores the memory of previous exposure. Our protocol can infer CRISPR-targeted sequences, including viruses, plasmids, and previously uncharacterized elements, and predict their hosts using unassembled short-read metagenomic sequencing data. By analyzing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences, which are likely complete circular genomes. The sequences included 2,154 tailed-phage genomes, together with 257 complete crAssphage genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 56 genomes of Inoviridae species, and 95 previously uncharacterized circular small genomes that have no reliably predicted protein-coding gene. We predicted the host(s) of approximately 70% of the discovered genomes at the taxonomic level of phylum by linking protospacers to taxonomically assigned CRISPR direct repeats. These results demonstrate that our protocol is efficient for de novo inference of CRISPR-targeted sequences and their host prediction.

Proposing a molecular classification associated with hypercoagulation in ovarian clear cell carcinoma.

BACKGROUND: Although ovarian clear cell carcinoma (CCC) is associated with high incidence of thromboembolism, the clinicopathological and biological significance of hypercoagulable status in CCC remains unclear. MATERIALS AND METHODS: We retrospectively analyzed pretreatment D-dimer levels, thromboembolic status, and clinical outcome of 125 CCCs in the discovery set and 143 CCCs in two other independent validation sets. Next, we performed RNA sequencing of 93 CCCs and compared coagulation-related gene profiles with 2492 pan-cancer data. We investigated differences in molecular characteristics of CCC subclasses based on coagulation status. RESULTS: In the discovery dataset, D-dimer elevation above the normal range was significantly associated with shorter progression-free and overall survival, irrespective to thromboembolic status. Multivariate analysis identified D-dimer elevation and clinical stage as an independent prognostic factors. We confirmed the prognostic significance of D-dimer elevation in the validation sets. Tissue factor and IL6, which are considered key elements of cancer-induced hypercoagulation, were highly expressed in CCC than in other cancers regardless of D-dimer level. Higher activity of various oncogenic pathways was observed in CCC with compared to without D-dimer elevation. Moreover, hierarchical cluster analysis divided 57 CCCs with D-dimer elevation into immunologically hot and cold tumor subtypes. Hot tumors were characterized by enrichment of T-cell inflamed phenotype, inflammation, the epithelial-mesenchymal transition, and high serum levels of CRP, and cold tumors by enrichment of cell cycle and MYC pathways. CONCLUSIONS: CCC represents hypercoagulable disease and elevate D-dimer is a prognostic factor for decreased survival in CCC. D-dimer high CCC has distinct molecular characteristics into the inflammatory-driven pathway (hot tumor) and the immune-suppressive pathway (cold tumor). Treatment implication of our proposed molecular classification merits further investigation.

Significance of Mitochondrial DNA Haplogroup on Epidermal Growth Factor Receptor Mutation in Japanese Patients With Lung Adenocarcinoma.

BACKGROUND/AIM: The frequency of somatic mutations of epidermal growth factor receptor (EGFR) in primary lung adenocarcinoma varies among populations and countries. The aim of the present study was to clarify whether the frequency of EGFR mutations in patients with lung adenocarcinoma depends on their mitochondrial DNA haplogroup, which reflects their maternal lineage. PATIENTS AND METHODS: Using normal lung tissue specimens, the mitochondrial DNA haplogroup was determined by multiplex polymerase chain reaction in 135 Japanese patients who underwent surgery for primary lung adenocarcinoma. RESULTS: The 135 patients were divided into two groups according to the two primitive haplotypes (N group, n=32; M group, n=103). The frequency of EGFR mutations in the N group was significantly higher than that in the M group (69% vs. 48%, p=0.044). The difference was prominent when the analysis was restricted to non-smokers (95% vs. 57%, p<0.01). CONCLUSION: The frequency of EGFR mutations in lung adenocarcinoma patients depends on their mitochondrial lineage.