Publications by authors named "Giang T Ong"

8 Publications

  • Page 1 of 1

Multiplex digital spatial profiling of proteins and RNA in fixed tissue.

Nat Biotechnol 2020 05 11;38(5):586-599. Epub 2020 May 11.

NanoString Technologies, Inc., Seattle, WA, USA.

Digital Spatial Profiling (DSP) is a method for highly multiplex spatial profiling of proteins or RNAs suitable for use on formalin-fixed, paraffin-embedded (FFPE) samples. The approach relies on (1) multiplexed readout of proteins or RNAs using oligonucleotide tags; (2) oligonucleotide tags attached to affinity reagents (antibodies or RNA probes) through a photocleavable (PC) linker; and (3) photocleaving light projected onto the tissue sample to release PC oligonucleotides in any spatial pattern across a region of interest (ROI) covering 1 to ~5,000 cells. DSP is capable of single-cell sensitivity within an ROI using the antibody readout, with RNA detection feasible down to ~600 individual mRNA transcripts. We show spatial profiling of up to 44 proteins and 96 genes (928 RNA probes) in lymphoid, colorectal tumor and autoimmune tissues by using the nCounter system and 1,412 genes (4,998 RNA probes) by using next-generation sequencing (NGS). DSP may be used to profile not only proteins and RNAs in biobanked samples but also immune markers in patient samples, with potential prognostic and predictive potential for clinical decision-making.
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http://dx.doi.org/10.1038/s41587-020-0472-9DOI Listing
May 2020

High-Plex Predictive Marker Discovery for Melanoma Immunotherapy-Treated Patients Using Digital Spatial Profiling.

Clin Cancer Res 2019 09 12;25(18):5503-5512. Epub 2019 Jun 12.

Department of Pathology, Yale School of Medicine, New Haven, Connecticut.

Purpose: Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma. The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables.

Results: The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response.

Conclusions: DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744974PMC
September 2019

A polyploid admixed origin of beer yeasts derived from European and Asian wine populations.

PLoS Biol 2019 03 5;17(3):e3000147. Epub 2019 Mar 5.

Pacific Northwest Research Institute, Seattle, Washington, United States of America.

Strains of Saccharomyces cerevisiae used to make beer, bread, and wine are genetically and phenotypically distinct from wild populations associated with trees. The origins of these domesticated populations are not always clear; human-associated migration and admixture with wild populations have had a strong impact on S. cerevisiae population structure. We examined the population genetic history of beer strains and found that ale strains and the S. cerevisiae portion of allotetraploid lager strains were derived from admixture between populations closely related to European grape wine strains and Asian rice wine strains. Similar to both lager and baking strains, ale strains are polyploid, providing them with a passive means of remaining isolated from other populations and providing us with a living relic of their ancestral hybridization. To reconstruct their polyploid origin, we phased the genomes of two ale strains and found ale haplotypes to both be recombinants between European and Asian alleles and to also contain novel alleles derived from extinct or as yet uncharacterized populations. We conclude that modern beer strains are the product of a historical melting pot of fermentation technology.
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http://dx.doi.org/10.1371/journal.pbio.3000147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400334PMC
March 2019

High-Throughput Identification of Adaptive Mutations in Experimentally Evolved Yeast Populations.

PLoS Genet 2016 Oct 11;12(10):e1006339. Epub 2016 Oct 11.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in Saccharomyces cerevisiae using sets of single-gene deletions and amplifications of almost all the genes in the S. cerevisiae genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously-but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution.
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http://dx.doi.org/10.1371/journal.pgen.1006339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065121PMC
October 2016

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

Genetics 2016 05 2;203(1):191-202. Epub 2016 Mar 2.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195 Department of Medicine, University of Washington, Seattle, Washington 98195 Howard Hughes Medical Institute, University of Washington Seattle, Washington 98195

In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach.
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http://dx.doi.org/10.1534/genetics.116.188037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858773PMC
May 2016

Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae.

Aging Cell 2016 Apr 13;15(2):317-24. Epub 2016 Jan 13.

Department of Genome Sciences, University of Washington, Foege Building, Room S403B, 3720 15th Ave NE, Box 355065, Seattle, WA, 98195-5065, USA.

Aneuploidy and aging are correlated; however, a causal link between these two phenomena has remained elusive. Here, we show that yeast disomic for a single native yeast chromosome generally have a decreased replicative lifespan. In addition, the extent of this lifespan deficit correlates with the size of the extra chromosome. We identified a mutation in BUL1 that rescues both the lifespan deficit and a protein trafficking defect in yeast disomic for chromosome 5. Bul1 is an E4 ubiquitin ligase adaptor involved in a protein quality control pathway that targets membrane proteins for endocytosis and destruction in the lysosomal vacuole, thereby maintaining protein homeostasis. Concurrent suppression of the aging and trafficking phenotypes suggests that disrupted membrane protein homeostasis in aneuploid yeast may contribute to their accelerated aging. The data reported here demonstrate that aneuploidy can impair protein homeostasis, shorten lifespan, and may contribute to age-associated phenotypes.
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http://dx.doi.org/10.1111/acel.12443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783355PMC
April 2016

The fitness consequences of aneuploidy are driven by condition-dependent gene effects.

PLoS Biol 2015 May 26;13(5):e1002155. Epub 2015 May 26.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell's proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp's fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.
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http://dx.doi.org/10.1371/journal.pbio.1002155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444335PMC
May 2015

The dynamics of diverse segmental amplifications in populations of Saccharomyces cerevisiae adapting to strong selection.

G3 (Bethesda) 2014 Mar 20;4(3):399-409. Epub 2014 Mar 20.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195.

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.
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http://dx.doi.org/10.1534/g3.113.009365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962480PMC
March 2014