The nexus of dynamic T cell states and immune checkpoint blockade therapy in the periphery and tumor microenvironment.

Immune checkpoint blockade (ICB) therapies, that is, using monoclonal antibodies to reinvigorate tumor-reactive, antigen-specific T cells from the inhibitory effects of CTLA-4, PD-1 and PD-L1 immune checkpoints, have revolutionized the therapeutic landscape of modern oncology. However, only a subset of patients can benefit from the ICB therapy. Biomarkers associated with ICB response, resistance and prognosis have been subjected to intensive research in the past decade. Early studies focused on the analysis of tumor specimens and their residing microenvironment. However, biopsies can be challenging to obtain in clinical practice, and do not reflect the dynamic changes of immunological parameters during the ICB therapy. Recent studies have investigated profiles of antigen-specific T cells derived from the peripheral compartment using multi-omics approaches. By tracking the clonotype and diversity of tumor-reactive T cell receptor repertoire, these studies collectively establish that de novo priming of antigen-specific T cells in peripheral blood occurs throughout the course of ICB, whereas preexisting T cells prior to ICB are exhausted to various degrees. Here, we review what is known about ICB-induced T cell phenotypic and functional changes in cancer patients both within the tumor microenvironment and in the peripheral compartment. A better understanding of parameters influencing the response to ICBs will provide rationales for developing novel diagnostics and combinatorial therapeutic strategies to maximize the clinical efficacies of ICB therapies.

[Peripheral Blood Laboratory Test Results Combined with TCF1+CD8+ T Lymphocytes 
Ratio to Predict the Response and Prognosis of Immunotherapy to 
Advanced Lung Cancer].

BACKGROUND: Immune checkpoint inhibitors (ICIs) therapy lacks viable biomarkers for response and prognosis prediction. This study aimed to investigate the correlation of peripheral blood laboratory test results combined with lymphocyte subset ratios to the response and prognosis of immunotherapy in advanced lung cancer. METHODS: Advanced lung cancer patients admitted to West China Hospital, Sichuan University from May 2021 to July 2023 were prospectively enrolled in this study. Clinical data and peripheral blood were collected before and after treatment and lymphocyte subset ratios were analyzed by flow cytometry. Logistic regression was used to identify factors correlated to ICIs treatment efficacy. Cox modeling was applied to explore the prognostic factors. RESULTS: Logistic regression showed that the baseline level of transcription factor T cell factor 1 (TCF1)+CD8+ T cell ratio and peripheral white blood cell (WBC) count, lymphocyte percentage, cytokeratin 19 fragment (CYFRA21-1) after 1 cycle of ICIs treatment were the potential predictors for ICIs response (P<0.05). Cox regression analysis showed that the baseline level of TCF1+CD8+ T cell ratio (P=0.020) and peripheral WBC count after 1 cycle of ICIs treatment (P<0.001) were prognostic factors. CONCLUSIONS: Patients with high baseline TCF1+CD8+ T cell ratio combined with low WBC counts and low CYFRA21-1 level after 1 cycle of ICIs treatment are more likely to benefit from ICIs therapy.

A novel cell-free DNA methylation-based model improves the early detection of colorectal cancer.

Screening for early-stage disease is vital for reducing colorectal cancer (CRC)-related mortality. Methylation of circulating tumor DNA has been previously used for various types of cancer screening. A novel cell-free DNA (cfDNA) methylation-based model which can improve the early detection of CRC is warranted. For our study, we collected 313 tissue and 577 plasma samples from patients with CRC, advanced adenoma (AA), non-AA and healthy controls. After quality control, 187 tissue DNA samples (91 non-malignant tissue from CRC patients, 26 AA and 70 CRC) and 489 plasma cfDNA samples were selected for targeted DNA methylation sequencing. We further developed a cfDNA methylation model based on 11 methylation biomarkers for CRC detection in the training cohort (area under curve [AUC] = 0.90 (0.85-0.94]) and verified the model in the validation cohort (AUC = 0.92 [0.88-0.96]). The cfDNA methylation model robustly detected patients pre-diagnosed with early-stage CRC (AUC = 0.90 [0.86-0.95]) or AA (AUC = 0.85 [0.78-0.91]). Here we established and validated a non-invasive cfDNA methylation model based on 11 DNA methylation biomarkers for the detection of early-stage CRC and AA. The utilization of the model in clinical practice may contribute to the early diagnosis of CRC.

Applications of Single-Cell Omics to Dissect Tumor Microenvironment.

The recent technical and computational advances in single-cell sequencing technologies have significantly broaden our toolkit to study tumor microenvironment (TME) directly from human specimens. The TME is the complex and dynamic ecosystem composed of multiple cell types, including tumor cells, immune cells, stromal cells, endothelial cells, and other non-cellular components such as the extracellular matrix and secreted signaling molecules. The great success on immune checkpoint blockade therapy has highlighted the importance of TME on anti-tumor immunity and has made it a prime target for further immunotherapy strategies. Applications of single-cell transcriptomics on studying TME has yielded unprecedented resolution of the cellular and molecular complexity of the TME, accelerating our understanding of the heterogeneity, plasticity, and complex cross-interaction between different cell types within the TME. In this review, we discuss the recent advances by single-cell sequencing on understanding the diversity of TME and its functional impact on tumor progression and immunotherapy response driven by single-cell sequencing. We primarily focus on the major immune cell types infiltrated in the human TME, including T cells, dendritic cells, and macrophages. We further discuss the limitations of the existing methodologies and the prospects on future studies utilizing single-cell multi-omics technologies. Since immune cells undergo continuous activation and differentiation within the TME in response to various environmental cues, we highlight the importance of integrating multimodal datasets to enable retrospective lineage tracing and epigenetic profiling of the tumor infiltrating immune cells. These novel technologies enable better characterization of the developmental lineages and differentiation states that are critical for the understanding of the underlying mechanisms driving the functional diversity of immune cells within the TME. We envision that with the continued accumulation of single-cell omics datasets, single-cell sequencing will become an indispensable aspect of the immune-oncology experimental toolkit. It will continue to drive the scientific innovations in precision immunotherapy and will be ultimately adopted by routine clinical practice in the foreseeable future.

Non-invasive diagnosis of early-stage lung cancer using high-throughput targeted DNA methylation sequencing of circulating tumor DNA (ctDNA).

Rational: LDCT screening can identify early-stage lung cancers yet introduces excessive false positives and it remains a great challenge to differentiate malignant tumors from benign solitary pulmonary nodules, which calls for better non-invasive diagnostic tools. Methods: We performed DNA methylation profiling by high throughput DNA bisulfite sequencing in tissue samples (nodule size < 3 cm in diameter) to learn methylation patterns that differentiate cancerous tumors from benign lesions. Then we filtered out methylation patterns exhibiting high background in circulating tumor DNA (ctDNA) and built an assay for plasma sample classification. Results: We first performed methylation profiling of 230 tissue samples to learn cancer-specific methylation patterns which achieved a sensitivity of 92.7% (88.3% - 97.1%) and a specificity of 92.8% (89.3% - 96.3%). These tissue-derived DNA methylation markers were further filtered using a training set of 66 plasma samples and 9 markers were selected to build a diagnostic prediction model. From an independent validation set of additional 66 plasma samples, this model obtained a sensitivity of 79.5% (63.5% - 90.7%) and a specificity of 85.2% (66.3% - 95.8%) for differentiating patients with malignant tumor (n = 39) from patients with benign lesions (n = 27). Additionally, when tested on gender and age matched asymptomatic normal individuals (n = 118), our model achieved a specificity of 93.2% (89.0% - 98.3%). Specifically, our assay is highly sensitive towards early-stage lung cancer, with a sensitivity of 75.0% (55.0%-90.0%) in 20 stage Ia lung cancer patients and 85.7% (57.1%-100.0%) in 7 stage Ib lung cancer patients. Conclusions: We have developed a novel sensitive blood based non-invasive diagnostic assay for detecting early stage lung cancer as well as differentiating lung cancers from benign pulmonary nodules.

Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer.

BACKGROUND: Analysis of cell-free DNA (cfDNA) is promising for broad applications in clinical settings, but with significant bias towards late-stage cancers. Although recent studies have discussed the diverse and degraded nature of cfDNA molecules, little is known about its impact on the practice of cfDNA analysis. METHODS: We developed single-strand library preparation and hybrid-capture-based cfDNA sequencing (SLHC-seq) to analysis degraded cfDNA fragments. Next we used SLHC-seq to perform cfDNA profiling in 112 pancreatic cancer patients, and the results were compared with 13 previous reports. Extensive analysis was performed in terms of cfDNA fragments to explore the reasons for higher detection rate of KRAS mutations in the circulation of pancreatic cancers. FINDINGS: By applying the new approach, we achieved higher efficiency in analysis of mutations than previously reported using other detection assays. 791 cancer-specific mutations were detected in plasma of 88% patients with KRAS hotspots detected in 70% of all patients. Only 8 mutations were detected in 28 healthy controls without any known oncogenic or truncating alleles. cfDNA profiling by SLHC-seq was largely consistent with results of tissue-based sequencing. SLHC-seq rescued short or damaged cfDNA fragments along to increase the sensitivity and accuracy of circulating-tumour DNA detection. INTERPRETATION: We found that the small mutant fragments are prevalent in early-stage patients, which provides strong evidence for fragment size-based detection of pancreatic cancer. The new pipeline enhanced our understanding of cfDNA biology and provide new insights for liquid biopsy.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03).Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.

Accessing Genetic Information with Liquid Biopsies.

Recent scientific advances in understanding circulating tumor cells, cell-free DNA/RNA, and exosomes in blood have laid a solid foundation for the development of routine molecular 'liquid biopsies'. This approach provides non-invasive access to genetic information--somatic mutations, epigenetic changes, and differential expression--about the physiological conditions of our body and diseases. It opens a valuable avenue for future genetic studies and human disease diagnosis, including prenatal and neurodegenerative disease diagnosis, as well as for cancer screening and monitoring. With the rapid development of highly sensitive and accurate technologies such as next-generation sequencing, molecular 'liquid biopsies' will quickly become a central piece in the future of precision medicine.

Somatic mutation in single human neurons tracks developmental and transcriptional history.

Neurons live for decades in a postmitotic state, their genomes susceptible to DNA damage. Here we survey the landscape of somatic single-nucleotide variants (SNVs) in the human brain. We identified thousands of somatic SNVs by single-cell sequencing of 36 neurons from the cerebral cortex of three normal individuals. Unlike germline and cancer SNVs, which are often caused by errors in DNA replication, neuronal mutations appear to reflect damage during active transcription. Somatic mutations create nested lineage trees, allowing them to be dated relative to developmental landmarks and revealing a polyclonal architecture of the human cerebral cortex. Thus, somatic mutations in the brain represent a durable and ongoing record of neuronal life history, from development through postmitotic function.

Cell lineage analysis in human brain using endogenous retroelements.

Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sublineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain.

Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.

CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain.

De novo copy-number variants (CNVs) can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS) in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥ 95%) neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG) due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ∼ 20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb) clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains.

Somatic mutation, genomic variation, and neurological disease.

Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one's parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease-even when present at low levels of mosaicism, for example-resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development.

Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain.

A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Because recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of three normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron, and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.

Somatic activation of AKT3 causes hemispheric developmental brain malformations.

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that two out of eight HMG samples showed trisomy of chromosome 1q, which encompasses many genes, including AKT3, a gene known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patient's blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralog in the brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to the brain could represent an important cause of complex neurogenetic disease.

Human mutations in NDE1 cause extreme microcephaly with lissencephaly [corrected].

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.

A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses.

In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.

A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication.

Most animals have two centrioles in spermatids (the distal and proximal centrioles), but insect spermatids seem to contain only one centriole (Fuller 1993), which functionally resembles the distal centriole. Using fluorescent centriolar markers, we identified a structure near the fly distal centriole that is reminiscent of a proximal centriole (i.e., proximal centriole-like, or PCL). We show that the PCL exhibits several features of daughter centrioles. First, a single PCL forms near the proximal segment of the older centriole. Second, the centriolar proteins SAS-6, Ana1, and Bld10p/Cep135 are in the PCL. Third, PCL formation depends on SAK/PLK4 and SAS-6. Using a genetic screen for PCL defect, we identified a mutation in the gene encoding the conserved centriolar protein POC1, which is part of the daughter centriole initiation site (Kilburn et al. 2007) in Tetrahymena. We conclude that the PCL resembles an early intermediate structure of a forming centriole, which may explain why no typical centriolar structure is observed under electron microscopy. We propose that, during the evolution of insects, the proximal centriole was simplified by eliminating the later steps in centriole assembly. The PCL may provide a unique model to study early steps of centriole formation.