We use cookies to understand how you use our site and to improve your experience. This includes personalizing content and advertising. To learn more, click here. By continuing to use our site, you accept our use of cookies. Cookie Policy.

Features Partner Sites Information LinkXpress
Sign In
Advertise with Us
INTEGRA BIOSCIENCES AG

Illumina

Illumina develops, manufactures and markets integrated systems for the analysis of genetic variations and biological ... read more Featured Products: More products

Download Mobile App




Events

09 Apr 2024 - 12 Apr 2024
15 Apr 2024 - 17 Apr 2024
23 Apr 2024 - 26 Apr 2024

Single-Cell Sequencing Reveals Clonal Diversity Among AML Patients

By LabMedica International staff writers
Posted on 03 Nov 2020
Print article
Image: The NovaSeq 6000 Sequencing System (Photo courtesy of Illumina).
Image: The NovaSeq 6000 Sequencing System (Photo courtesy of Illumina).
A growing body of evidence supports the role of clonal diversity in therapeutic resistance, recurrence, and poor outcomes in cancer. Clonal diversity also reflects the history of the accumulation of somatic mutations within a tumor.

The ability to infer clonal heterogeneity and tumor phylogeny from bulk sequencing data is inherently limited, because bulk sequencing techniques cannot reliably infer mutation co-occurrences and hence often fail in accurately reconstructing clonal substructure. Single-cell DNA sequencing (scDNA-seq) can address some of these challenges.

A large team of scientists at the University of Texas MD Anderson Cancer Center (Houston, TX, USA) analyzed 154 samples (140 bone marrow mononuclear cells (BMMCs) and 14 peripheral blood mononuclear cells) from 123 patients with acute myeloid leukemia (AML) who had at least one somatic mutation covered by the targeted panel for scDNA-seq. Of the 123 patients, 108 patients were analyzed for the single-timepoint sample collected at pre-treatment (N = 98) or relapsed/refractory timepoint (N = 10). Among 123 patients, 97 were analyzed by scDNA-seq, 23 were analyzed by the simultaneous single-cell DNA and cell surface protein sequencing (scDNA+protein-seq), and three were analyzed by scDNA-seq and scDNA+protein-seq.

The pooled library was sequenced by one of the following sequencing platforms, MiSeq, HiSeq 4000, or NovaSeq 6000 (Illumina, Sand Diego, CA, USA) with 150- or 250-base pair (bp) paired-end multiplexed runs. The team performed droplet digital PCR (ddPCR) using QX200 Droplet Digital System (Bio-Rad Laboratories, Hercules, CA, USA) to confirm the variants that were detected by scDNA-seq, but were not detected by bulk-seq. Simultaneous profiling of DNA mutation and cell-surface immunophenotype (scDNA+protein-seq) was performed using the custom-designed panel kit and 10–15 oligo-conjugated antibodies (Mission Bio, South San Francisco, CA, USA). Immunophenotypes of the bone marrow cells from AML patients were assessed using eight-color flow cytometry on FACSCanto II (BD Biosciences, San Jose, CA, USA).

In all, the scientists sequenced more than 730,000 cells to find 543 somatic mutations in 31 cancer-related genes, 98% of which they orthogonally validated. The most common mutations they detected were in NPM1, followed by ones in DNMT3A and NRAS. They further found that while a number of mutations that were functionally redundant were found in the same patients, the alterations were often found in mutually exclusive clones. This extended to alterations affecting receptor tyrosine kinase (RTK)/Gas GTPase (RAS)/MAP kinase (MAPK) signaling pathway genes as well as IDH1 and IDH2 mutations and TET2 and IDH mutations. This suggested to the scientists that cells either do not need two mutations or that, when they appear together, the mutations are toxic, which could suggest a potential treatment avenue to investigate.

The investigators also analyzed genotype-phenotype correlations among the cells to find, for instance, that cells with NPM1 or IDH mutations expressed lower levels of CD34 and HLA-DR, while cells with a single TP53 mutations had CD34+CD117+ phenotype, but double TP53 mutations had a monocytic immunophenotype.

Koichi Takahashi, MD, PhD, the senior author of the study, said, “This information is also somewhat available from longitudinal bulk sequencing data longitudinally, but I think single-cell data uniquely provides this meticulous view of clone-by-clone dynamics, which is just simply not possible by bulk sequencing.” The study was published on October 21, 2020 in the journal Nature Communications.


Platinum Member
COVID-19 Rapid Test
OSOM COVID-19 Antigen Rapid Test
One Step HbA1c Measuring System
GREENCARE A1c
Complement 3 (C3) Test
GPP-100 C3 Kit
New
Gold Member
TORCH Panel Rapid Test
Rapid TORCH Panel Test

Print article

Channels

Clinical Chemistry

view channel
Image: Reaching speeds up to 6,000 RPM, this centrifuge forms the basis for a new type of inexpensive, POC biomedical test (Photo courtesy of Duke University)

POC Biomedical Test Spins Water Droplet Using Sound Waves for Cancer Detection

Exosomes, tiny cellular bioparticles carrying a specific set of proteins, lipids, and genetic materials, play a crucial role in cell communication and hold promise for non-invasive diagnostics.... Read more

Hematology

view channel
Image: The low-cost portable device rapidly identifies chemotherapy patients at risk of sepsis (Photo courtesy of 52North Health)

POC Finger-Prick Blood Test Determines Risk of Neutropenic Sepsis in Patients Undergoing Chemotherapy

Neutropenia, a decrease in neutrophils (a type of white blood cell crucial for fighting infections), is a frequent side effect of certain cancer treatments. This condition elevates the risk of infections,... Read more

Pathology

view channel
Image: The OvaCis Rapid Test discriminates benign from malignant epithelial ovarian cysts (Photo courtesy of INEX)

Intra-Operative POC Device Distinguishes Between Benign and Malignant Ovarian Cysts within 15 Minutes

Ovarian cysts represent a significant health issue for women globally, with up to 10% experiencing this condition at some point in their lives. These cysts form when fluid collects within a thin membrane... Read more
Copyright © 2000-2024 Globetech Media. All rights reserved.