Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and is well-suited for the development of affordable multiplex protein assays, which provides the potential to accelerate proteomics research.

Keratin 15, transcobalamin I and homeobox gene Hox-B13 expression in breast phyllodes tumors: novel markers in biological classification.

Breast phyllodes tumors are rare neoplasms which present challenges for histological classification. Microscopic features are not always predictive of clinical behavior, and scarce data exist on the prognostic role of biological markers. Our study evaluated a series of 145 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital between 2006 and 2009, incorporating 91 (62.8%) benign, 40 (27.6%) borderline, and 14 (9.7%) malignant phyllodes tumors. Antibodies to keratin 15 (KRT15), transcobalamin I (TCN1), and homeobox gene Hox-B13 (HOXB13) were applied to sections cut from tissue microarray blocks. KRT15 and TCN1 positivity was defined when there was reactivity of 1% or more stromal cells, while HOXB13 positivity was defined using a H-score of 100 and above. Positive immunohistochemical expression for KRT15, TCN1, and HOXB13 was seen in 21 (14.5%), 96 (66.2%), and 66 (45.5%) of tumors, respectively. Stromal expression of KRT15, TCN1, and HOXB13 was significantly correlated with tumor grade (P < 0.001, P < 0.001, P = 0.012), stromal hypercellularity (P = 0.005, P < 0.001, P = 0.023), mitotic activity (P < 0.001), and microscopic borders (P = 0.006, P < 0.001, P = 0.011). Co-expression of TCN1 and HOXB13 was seen in 21 of 91 (23.1%) benign, 18 of 40 (45.0%) borderline, and 11 of 14 (78.6%) malignant tumors, suggesting that the dual-marker panels of TCN1 and HOXB13 might be helpful in classifying borderline and malignant tumors. Although expression of TCN1 alone was present in all malignant and 34 of 40 (85.0%) borderline tumors, a combined panel with HOXB13 excluded some benign cases and was a better discriminant for a significant proportion of borderline and malignant tumors.

Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis.

BACKGROUND: High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features. METHODS: This study was based on 65 patients with SCC tongue who underwent primary surgical treatment including neck dissection. The patients were categorized into 2 groups based on the presence of malignancy in their cervical lymph nodes. A review of the patients' pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients' clinical and pathological features. RESULTS: The total number of HEV was found to be significantly associated to disease-free interval. There was a similar association comparing the HEV parameters to overall survival. The density of abnormal HEV was significantly higher in patients with established metastases in their lymph nodes and HEV was shown to be a better prognosis factor than conventional tumor staging. The HEV morphological metamorphosis demonstrates a spectrum that correlates well with disease progression and clinical outcome. CONCLUSIONS: The results suggest that the HEV displays a spectrum of morphological changes in the presence of cancer and LN metastasis, and that HEV is possibly involved in the process of cancer metastasis. We revealed the relationship of HEV and their metamorphosis in pre-metastatic and metastatic environments in regional lymph nodes of tongue cancer patients in relation to clinical outcomes. The significant observation of modified dilated HEV containing red blood cells in lymph nodal basin of a cancer suggests the shifting of its function from one primarily of immune response to that of a blood carrying vessel. It also demonstrated potential prognostic value. More studies are needed to elucidate its potential role in cancer immunotherapy and as a potential novel therapeutic approach to preventing metastasis by manipulating the remodelling processes of HEV.

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.

Simultaneous DNA, RNA, and Protein Analysis from Single Cells Using a High-Throughput Microfluidic Workflow for Resolution of Genotype-to-Phenotype Modalities.

Understanding the genomic landscape of cancer in single cells can be valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using microfluidics paired with next-generation sequencing. Our procedure determines targeted protein levels, mRNA transcript levels, and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts, and DNA amplicons.

High-Throughput Multimodal Single-Cell Targeted DNA and Surface Protein Analysis Using the Mission Bio Tapestri Platform.

An important aspect of understanding cancer biology is to connect the diverse repertoire of genotype-to-phenotype displays in individual specimens and ultimately resolve disease course outcome through informative datasets. A focus of cancer genomics has strived to provide predictive capabilities using genomic information to further inform therapeutic strategies. The advent of single-cell sequencing and analysis now provides a route to decipher high-resolution genomic diversity in individual samples and facilitate detailed understanding of clonal evolution in clinical research settings. In addition to generating high-throughput single-cell genomic SNV and CNV data, this protocol describes a new analytical ability that adds a second dimension which provides for interrogation of surface protein marker expression. The first immediate application of this technology is quite suitable to heme cancer cell studies. This multimodal approach allows for correlation of diverse genomic signatures to key phenotypic biomarkers such as immunophenotypes in leukemic diseases.

Molecular classification of breast phyllodes tumors: validation of the histologic grading scheme and insights into malignant progression.

Phyllodes tumors of the breast are rare fibroepithelial neoplasms with a potential for recurrence. Current histological classification is not always predictive of clinical behavior. The aim of this study was to identify genetic changes associated with the development of borderline and malignant phyllodes tumors in an Asian population, and to assess if genetic data supported the categorization of these tumors into the existing three grades of benign, borderline, and malignant. Expression profiling of 21 phyllodes tumors (6 benign, 10 borderline, 5 malignant) was performed using Affymetrix U133Plus 2.0 GeneChips(®). Gene expression among benign, borderline, and malignant tumors was compared and a 29 gene list was able to classify them according to their histologic grade. Among these 29 genes are those responsible for matrix formation, cell adhesion, epidermis formation, and cell proliferation. Comparative genomic microarray analysis showed that the most common chromosomal alteration associated with borderline and malignant tumors was 1q gain, and an increasing number of chromosomal changes was noted with increasing histological grade. Upregulation of HOXB13 was seen in malignant relative to borderline phyllodes tumors and further investigated by immunohistochemistry in a corresponding set of formalin-fixed, paraffin-embedded tumors. HOXB13 protein overexpression was found to be correlated with stromal hypercellularity and atypia (P = 0.03, P = 0.039, respectively) and may be implicated in the development of malignant phyllodes tumors.

Personalized Single-Cell Proteogenomics to Distinguish Acute Myeloid Leukemia from Non-Malignant Clonal Hematopoiesis.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.

Benchmarking single-cell RNA-sequencing protocols for cell atlas projects.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.

Virus-specific read-through codon preference affects infectivity of chimeric cucumber green mottle mosaic viruses displaying a dengue virus epitope.

A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.

Single-Cell Dosing and mRNA Sequencing of Suspension and Adherent Cells Using the PolarisTM System.

Single-cell functional analysis provides a natural next step in the now widely adopted single-cell mRNA sequencing studies. Functional studies can be designed to study cellular context by using single-cell culture, perturbation, manipulation, or treatment. Here we present a method for a functional study of 48 single cells by single-cell isolation, dosing, and mRNA sequencing with an integrated fluidic circuit (IFC) on the Fluidigm® Polaris™ system. The major procedures required to execute this protocol are (1) cell preparation and staining; (2) priming, single-cell selection, cell dosing, cell staining, and cDNA generation on the Polaris IFC; and (3) preparation and sequencing of single-cell mRNA-seq libraries. The cell preparation and staining steps employ the use of a universal tracking dye to trace all cells that enter the IFC, while additional fluorescence dyes chosen by the user can be used to differentiate cell types in the overall mix. The steps on the Polaris IFC follow standard protocols, which are also described in the Fluidigm user documentation. The library preparation step adds Illumina® Nextera® XT indexes to the cDNA generated on the Polaris IFC. The resulting sequencing libraries can be sequenced on any Illumina sequencing platform.

Simultaneous Targeted Detection of Proteins and RNAs in Single Cells.

Simultaneous detection of both RNA and protein in individual single cells offers a powerful tool for genotype-to-phenotype investigations. Proximity extension assay (PEA) is a quantitative, sensitive, and multiplex protein detection system that has superb utility in single-cell omic analysis. We implemented PEA using the flexible microfluidic workflow of the Fluidigm® C1™ system followed by real-time quantitative polymerase chain reaction (RT-qPCR) on the Fluidigm Biomark™ HD system. With this workflow, targeted quantification of RNAs and proteins within individual cells is readily conducted.

Silencing the Snail-Dependent RNA Splice Regulator ESRP1 Drives Malignant Transformation of Human Pulmonary Epithelial Cells.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR.

Single-cell protein-mRNA correlation analysis enabled by multiplexed dual-analyte co-detection.

We have investigated the correlation between proteins and mRNAs in single cells employing an integrated workflow for dual-analyte co-detection. This is achieved by combining the oligo extension reaction (OER), which converts protein levels to DNA levels, with reverse transcription for mRNA detection. Unsupervised gene expression profiling analysis, including principal component analysis and hierarchical clustering, revealed different aspects of the protein-mRNA relationship. Violin plot analysis showed that some genes exhibited similar distribution patterns for proteins and mRNAs. We also demonstrate that cells can be separated into subpopulations based on their protein-mRNA expression profiles, and that different subpopulations have distinct correlation coefficient values. Our results demonstrated that integrated investigations of mRNA and protein levels in single cells allows comprehensive analysis not attainable at bulk levels.

Corrigendum: Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

[This corrects the article on p. 70 in vol. 4, PMID: 27709111.].

Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics.

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.

Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.

Molecular Pathways: Targeting Cellular Energy Metabolism in Cancer via Inhibition of SLC2A1 and LDHA.

Reprogramming of cellular energy metabolism is widely accepted to be one of the main hallmarks of cancer. The aberrant expression pattern of key regulators in the glycolysis pathway in cancer cells corroborates with the hypothesis that most cancer cells utilize aerobic glycolysis as their main ATP production method instead of mitochondrial oxidative phosphorylation. Overexpression of SLC2A1 and LDHA, both important regulators of the glycolysis pathway, was detected in the premalignant lesions and tumors of lung cancer patients, suggesting the involvement of these proteins in early carcinogenesis and tumor progression in cancer. Preclinical studies demonstrated that inhibiting SLC2A1 or LDHA led to diminished tumor growth in vitro and in vivo. SLC2A1 and LDHA inhibitors, when administered in combination with other chemotherapeutic agents, showed synergistic antitumor effects by resensitizing chemoresistant cancer cells to the chemotherapies. These results indicate that disrupting SLC2A1, LDHA, or other regulators in cancer cell energetics is a very promising approach for new targeted therapies.

Potential genetic anticipation in hereditary leiomyomatosis-renal cell cancer (HLRCC).

Hereditary leiomyomatosis-renal cell cancer (HLRCC) is an autosomal dominant disorder characterised by cutaneous leiomyomas, symptomatic uterine leiomyomas and aggressive type II papillary renal cell carcinoma. It is caused by heterozygous mutations in the fumarate hydratase (FH) gene on chromosome 1q43. We present evidence of genetic anticipation in HLRCC syndrome. A comprehensive literature review was performed to determine the potential for genetic anticipation in HLRCC syndrome. The normal random effects model was used to evaluate for genetic anticipation to ensure reduction in bias. A total of 11 FH kindreds with available multi-generational data were identified for analysis. The mean difference in age at diagnosis of RCC between the first and second generation was -18.6 years (95 % CI -26.6 to -10.6, p < 0.001). The mean difference in age at diagnosis of RCC between the first and third generation was -36.2 years (95 % CI -47.0 to -25.4, p < 0.001). No evidence of anticipation for uterine leiomyomas was observed (p = 0.349). We report preliminary evidence of genetic anticipation of RCC in HLRCC syndrome. Additional clinical validation is important to confirm this observation, which may have practical implications on counseling and timing of surveillance initiation. Exploration of the underlying mechanisms of anticipation in HLRCC would be of considerable biological interest.