Longitudinal Analyses of Mutational Subclonal Architecture and Tumor Subtypes in Recurrent Bladder Cancer.

Longitudinal tumor sequencing of recurrent bladder cancer (BC) can facilitate the investigation of BC progression-associated genomic and transcriptomic alterations. In this study, we analyzed 18 tumor specimens including distant and locoregional metastases obtained during tumor progression for five BC patients using whole-exome and transcriptome sequencing. Along with the substantial level of intratumoral mutational heterogeneity across the cases, we observed that clonal mutations were enriched with known BC driver genes and apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC)-associated mutation signatures compared with subclonal mutations, suggesting the genetic makeup for BC tumorigenesis associated with APOBEC deaminase activity was accomplished early in the cancer evolution. Mutation-based phylogenetic analyses also revealed temporal dynamics of mutational clonal architectures in which the number of mutational clones varied along the BC progression and notably was often punctuated by clonal sweeps associated with chemotherapy. The bulk-level transcriptome sequencing revealed frequent subtype switching in which transcriptionally defined BC subtypes may vary during tumor progression. Longitudinal whole-exome and transcriptome sequencing of recurrent BC may advance our understanding into the BC heterogeneity in terms of somatic mutations, cell clones and transcriptome-based tumor subtypes during disease progression.

Provably Secure Mutual Authentication and Key Agreement Scheme Using PUF in Internet of Drones Deployments.

Internet of Drones (IoD), designed to coordinate the access of unmanned aerial vehicles (UAVs), is a specific application of the Internet of Things (IoT). Drones are used to control airspace and offer services such as rescue, traffic surveillance, environmental monitoring, delivery and so on. However, IoD continues to suffer from privacy and security issues. Firstly, messages are transmitted over public channels in IoD environments, which compromises data security. Further, sensitive data can also be extracted from stolen mobile devices of remote users. Moreover, drones are susceptible to physical capture and manipulation by adversaries, which are called drone capture attacks. Thus, the development of a secure and lightweight authentication scheme is essential to overcoming these security vulnerabilities, even on resource-constrained drones. In 2021, Akram et al. proposed a secure and lightweight user-drone authentication scheme for drone networks. However, we discovered that Akram et al.'s scheme is susceptible to user and drone impersonation, verification table leakage, and denial of service (DoS) attacks. Furthermore, their scheme cannot provide perfect forward secrecy. To overcome the aforementioned security vulnerabilities, we propose a secure mutual authentication and key agreement scheme between user and drone pairs. The proposed scheme utilizes physical unclonable function (PUF) to give drones uniqueness and resistance against drone stolen attacks. Moreover, the proposed scheme uses a fuzzy extractor to utilize the biometrics of users as secret parameters. We analyze the security of the proposed scheme using informal security analysis, Burrows-Abadi-Needham (BAN) logic, a Real-or-Random (RoR) model, and Automated Verification of Internet Security Protocols and Applications (AVISPA) simulation. We also compared the security features and performance of the proposed scheme and the existing related schemes. Therefore, we demonstrate that the proposed scheme is suitable for IoD environments that can provide users with secure and convenient wireless communications.

Transcriptional Profiling Reveals Mesenchymal Subtypes of Small Cell Lung Cancer with Activation of the Epithelial-to-Mesenchymal Transition and Worse Clinical Outcomes.

While molecular subtypes of small cell lung cancers (SCLC) based on neuroendocrine (NE) and non-NE transcriptional regulators have been established, the association between these molecular subtypes and recently recognized SCLC-inflamed (SCLC-I) tumors is less understood. In this study, we used gene expression profiles of SCLC primary tumors and cell lines to discover and characterize SCLC-M (mesenchymal) tumors distinct from SCLC-I tumors for molecular features, clinical outcomes, and cross-species developmental trajectories. SCLC-M tumors show elevated epithelial-to-mesenchymal transformation (EMT) and YAP1 activity but a low level of anticancer immune activity and worse clinical outcomes than SCLC-I tumors. The prevalence of SCLC-M tumors was 3.2-7.4% in primary SCLC cohorts, which was further confirmed by immunohistochemistry in an independent cohort. Deconvoluted gene expression of tumor epithelial cells showed that EMT and increased immune function are tumor-intrinsic characteristics of SCLC-M and SCLC-I subtypes, respectively. Cross-species analysis revealed that human primary SCLC tumors recapitulate the NE-to-non-NE progression murine model providing insight into the developmental relationships among SCLC subtypes, e.g., early NE (SCLC-A and -N)- vs. late non-NE tumors (SCLC-M and -P). Newly identified SCLC-M tumors are biologically and clinically distinct from SCLC-I tumors which should be taken into account for the diagnosis and treatment of the disease.

Spatially Distinct Reprogramming of the Tumor Microenvironment Based On Tumor Invasion in Diffuse-Type Gastric Cancers.

PURPOSE: Histologic features of diffuse-type gastric cancer indicate that the tumor microenvironment (TME) may substantially impact tumor invasiveness. However, cellular components and molecular features associated with cancer invasiveness in the TME of diffuse-type gastric cancers are poorly understood. EXPERIMENTAL DESIGN: We performed single-cell RNA-sequencing (scRNA-seq) using tissue samples from superficial and deep invasive layers of cancerous and paired normal tissues freshly harvested from five patients with diffuse-type gastric cancer. The scRNA-seq results were validated by immunohistochemistry (IHC) and duplex in situ hybridization (ISH) in formalin-fixed paraffin-embedded tissues. RESULTS: Seven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type-specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulated CCL2 transcripts. IHC and duplex ISH revealed the distribution of the major cell types and CCL2-expressing endothelial cells and fibroblasts, indicating tumor invasion. Elevation of CCL2 levels along the superficial-to-deep layer axis revealed the immunosuppressive immune cell subtypes that may contribute to tumor cell aggressiveness in the deep invasive layers of diffuse-type gastric cancer. The analyses of public datasets revealed the high-level coexpression of stromal cell-specific genes and that CCL2 correlated with poor survival outcomes in patients with gastric cancer. CONCLUSIONS: This study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type gastric cancer. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type gastric cancer.See related commentary by Huang and Brekken, p. 6284.

Exosomal miR-1305 in the oncogenic activity of hypoxic multiple myeloma cells: a biomarker for predicting prognosis.

Background: Exosomes have emerged as important mediators of tumor progression, and a prognostic role for serum exosomal miRNAs has been suggested in multiple myeloma (MM). Given the association of hypoxia with tumor aggressiveness, including cancer stem cell-like phenotypes, we explored exosomal miRNAs from MM cells under hypoxic conditions and analyzed their diverse roles both in promoting oncogenic activity and in predicting prognosis. Methods: The human MM cell line, RPMI 8226, was cultured under hypoxic conditions and their exosome production and exosomal miRNA profiles were compared with those of normoxic parental cells. The survival outcome of myeloma patients was compared using serum levels of exosomal miRNAs, and the effects of exosomal miRNAs on the target genes of MM cells and adjacent immune cells were analyzed. Results: Increased expression of stem cell markers and exosome production were observed in hypoxic MM cells. Exosome miRNA analysis identified a higher expression of miR-1305 in exosomes isolated from hypoxic MM cells than in those of normoxic parental cells. The overall survival of patients with high exosomal miR-1305 was poorer than it was in patients with low exosomal miR-1305. In hypoxic MM cells, an increase of exosomal miR-1305 led to a decrease of cellular miR-1305 and increased expression of the miR-1305 target genes, MDM2, IGF1 and FGF2 resulted in the promotion of oncogenic activity of MM. Exosomal miR-1305 was also transferred from MM cells to macrophages, and miR-1305-transferred macrophages showed tumor-promoting, M2-macrophage phenotypes. Conclusions: Exosome-mediated secretion of miR-1305 in MM cells promoted oncogenic activity of hypoxic MM cells and high serum levels of exosomal miR-1305.

Tumor-promoting macrophages prevail in malignant ascites of advanced gastric cancer.

Gastric cancer (GC) patients develop malignant ascites as the disease progresses owing to peritoneal metastasis. GC patients with malignant ascites have a rapidly deteriorating clinical course with short survival following the onset of malignant ascites. Better optimized treatment strategies for this subset of patients are needed. To define the cellular characteristics of malignant ascites of GC, we used single-cell RNA sequencing to characterize tumor cells and tumor-associated macrophages (TAMs) from four samples of malignant ascites and one sample of cerebrospinal fluid. Reference transcriptomes for M1 and M2 macrophages were generated by in vitro differentiation of healthy blood-derived monocytes and applied to assess the inflammatory properties of TAMs. We analyzed 180 cells, including tumor cells, macrophages, and mesothelial cells. Dynamic exchange of tumor-promoting signals, including the CCL3-CCR1 or IL1B-IL1R2 interactions, suggests macrophage recruitment and anti-inflammatory tuning by tumor cells. By comparing these data with reference transcriptomes for M1-type and M2-type macrophages, we found noninflammatory characteristics in macrophages recovered from the malignant ascites of GC. Using public datasets, we demonstrated that the single-cell transcriptome-driven M2-specific signature was associated with poor prognosis in GC. Our data indicate that the anti-inflammatory characteristics of TAMs are controlled by tumor cells and present implications for treatment strategies for GC patients in which combination treatment targeting cancer cells and macrophages may have a reciprocal synergistic effect.

Gene expression signatures associated with sensitivity to azacitidine in myelodysplastic syndromes.

Allogeneic stem cell transplantation is currently the only curative treatment option for myelodysplastic syndromes (MDS). Pre-transplant debulking treatment have been employed for advanced MDS and we previously reported that marrow response (blast ≤ 5%) following the bridging therapy with hypomethylating agent was an independent favorable factor for survival; however, it is still not clear which patients will respond to hypomethylating agent and which genomic features can predict the response. In this study, we performed RNAseq for 23 MDS patients among which 14 (61%) and 9 (39%) patients showed marrow complete remission and primary resistance to azacitidine, respectively. Differential expression-based analyses of treatment-naive, baseline gene expression profiles revealed that molecular functions representing mitochondria and apoptosis were up-regulated in responders. In contrast, we identified genes involved in the Wnt pathway were relatively up-regulated in non-responders. In independent validation cohorts of MDS patients, the expression of gene sets specific to non-responders and responders distinguished the patients with favorable prognosis and those responded to azacitidine highlighting the prognostic and predictive implication. In addition, a systems biology approach identified genes involved in ubiquitination, such as UBC and PFDN2, which may be key players in the regulation of differential gene expression in treatment responders and non-responders. Taken together, identifying the gene expression signature may advance our understanding of the molecular mechanisms of azacitidine and may also serve to predict patient responses to drug treatment.

Genomic, transcriptomic, and viral integration profiles associated with recurrent/metastatic progression in high-risk human papillomavirus cervical carcinomas.

Acquisition of recurrent/metastatic potential by a tumor cell defines a critical step in malignant progression. However, understanding of metastatic progression at the molecular level is scarce for cervical carcinomas (CES). In this study, we performed genomic, transcriptomic, and viral profiling of five pairs of primary (CES-P) and matched recurrent/metastatic tumors (CES-R/M) with high risk human papillomavirus. Whole exome sequencing revealed mutation features of CES-R/M including elevated mutation burdens and prevalent copy number alterations compared to their matched CES-P. A relative deficit of APOBEC-related mutation signatures accompanying the transcriptional downregulation of APOBEC3A was observed for CES-R/M. Mutations in genes encoding epigenetic regulators were commonly observed as CES-R/M-specific alterations. Immunoprofiling and gene set analysis revealed CES-Ps were enriched with transcripts representing activated anticancer immunity such as interferon-gamma pathway, while CES-R/M exhibited upregulation of genes involved in epithelial-mesenchymal transition and angiogenesis. Viral capture sequencing revealed that integration sites remained enriched in viral E1 protein domain during malignant progression. Moreover, we found transcriptional upregulation of POSTN and downregulation of APOBEC3A were associated with unfavorable clinical outcomes in CES. Comprehensive genomic and transcriptomic profiling of a rare cohort including CES-R/M identified metastases-specific features to advance the molecular understanding into CES metastatic progression with potential clinical implications.

Earlier-Phased Cancer Immunity Cycle Strongly Influences Cancer Immunity in Operable Never-Smoker Lung Adenocarcinoma.

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.

Alterations in the Transcriptional Programs of Myeloma Cells and the Microenvironment during Extramedullary Progression Affect Proliferation and Immune Evasion.

PURPOSE: In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites. EXPERIMENTAL DESIGN: Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed. RESULTS: Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells. CONCLUSIONS: These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion.

Junction Location Identifier (JuLI): Accurate Detection of DNA Fusions in Clinical Sequencing for Precision Oncology.

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

Paired whole exome and transcriptome analyses for the Immunogenomic changes during concurrent chemoradiotherapy in esophageal squamous cell carcinoma.

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.

Biomarkers Associated with Tumor Heterogeneity in Prostate Cancer.

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.

Correction: The mutational landscape of ocular marginal zone lymphoma identifies frequent alterations in TNFAIP3 followed by mutations in TBL1XR1 and CREBBP.

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

Linking transcriptional and genetic tumor heterogeneity through allele analysis of single-cell RNA-seq data.

Characterization of intratumoral heterogeneity is critical to cancer therapy, as the presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. Although genetic variation is a well-studied source of intratumoral heterogeneity, the functional impact of most genetic alterations remains unclear. Even less understood is the relative importance of other factors influencing heterogeneity, such as epigenetic state or tumor microenvironment. To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss of heterozygosity in individual cells from single-cell RNA-sequencing data. By integrating allele and normalized expression information, HoneyBADGER is able to identify and infer the presence of subclone-specific alterations in individual cells and reconstruct the underlying subclonal architecture. By examining several tumor types, we show that HoneyBADGER is effective at identifying deletions, amplifications, and copy-neutral loss-of-heterozygosity events and is capable of robustly identifying subclonal focal alterations as small as 10 megabases. We further apply HoneyBADGER to analyze single cells from a progressive multiple myeloma patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression. Other prominent transcriptional subpopulations within these tumors did not line up with the genetic subclonal structure and were likely driven by alternative, nonclonal mechanisms. These results highlight the need for integrative analysis to understand the molecular and phenotypic heterogeneity in cancer.

Prevalence and detection of low-allele-fraction variants in clinical cancer samples.

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.

The mutational landscape of ocular marginal zone lymphoma identifies frequent alterations in TNFAIP3 followed by mutations in TBL1XR1 and CREBBP.

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.

Comprehensive genomic profiling of IgM multiple myeloma identifies IRF4 as a prognostic marker.

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.

Deciphering intratumor heterogeneity using cancer genome analysis.

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