Targeted, Amplicon-Based, Next-Generation Sequencing to Detect Age-Related Clonal Hematopoiesis

2019 ◽  
pp. 167-180 ◽  
Author(s):  
Brooke Snetsinger ◽  
Christina K. Ferrone ◽  
Michael J. Rauh
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
Age Related ◽  
Generation Sequencing

scholarly journals Clonal Hematopoiesis From Next Generation Sequencing of Plasma From a Patient With Lung Adenocarcinoma: A Case Report

2020 ◽  
Vol 10 ◽  
Author(s):  
Munehiro Ito ◽  
Yutaka Fujiwara ◽  
Takashi Kubo ◽  
Hiromichi Matsushita ◽  
Tadashi Kumamoto ◽  
...  
Keyword(s):  
Case Report ◽  
Next Generation Sequencing ◽  
Lung Adenocarcinoma ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
Generation Sequencing

scholarly journals Transcriptomic analysis highlights cochlear inflammation associated with age-related hearing loss in C57BL/6 mice using next generation sequencing

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9737
Author(s):  
Zhongwu Su ◽  
Hao Xiong ◽  
Yi Liu ◽  
Jiaqi Pang ◽  
Hanqing Lin ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Molecular Mechanisms ◽  
Transcriptomic Analysis ◽  
Differentially Expressed ◽  
Next Generation ◽  
Age Related ◽  
Cochlear Inflammation ◽  
Age Related Hearing Loss ◽  
Generation Sequencing

Background In our aging society, age-related hearing loss (AHL) is the most common sensory disorder in old people. Much progress has been made in understanding the pathological process of AHL over the past few decades. However, the mechanism of cochlear degeneration during aging is still not fully understood. Methods Next generation sequencing technique was used to sequence the whole transcriptome of the cochlea of C57BL/6 mice, a mouse model of AHL. Differentially expressed genes (DEGs) were identified using the Cuffdiff software. GO and KEGG pathway enrichment analyses of the DEGs were implemented by using the GOseq R package and KOBAS software, respectively. Results A total of 731 genes (379 up- and 352 down-regulated) were revealed to be differentially expressed in the cochlea of aged mice compared to the young. Many genes associated with aging, apoptosis, necroptosis and particularly, inflammation were identified as being significantly modulated in the aged cochlea. GO and KEGG analyses of the upregulated DEGs revealed that the most enriched terms were associated with immune responses and inflammatory pathways, whereas many of the downregulated genes are involved in ion channel function and neuronal signaling. Real-time qPCR showed that H2O2 treatment significantly induced the expression of multiple inflammation and necroptosis-related genes in HEI-OC1 cells. Conclusion Using next generation sequencing, our transcriptomic analysis revealed the differences of gene expression pattern with age in the cochlea of C57BL/6 mice. Our study also revealed multiple immune and inflammatory transcriptomic changes during cochlear aging and provides new insights into the molecular mechanisms underlying cochlear inflammation in AHL.

Evaluation of clonal hematopoiesis in late stage NSCLC using a next-generation sequencing panel targeting cancer genes.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 9050-9050 ◽  
Author(s):  
Stephanie J. Yaung ◽  
Frederike Fuhlbrück ◽  
Johnny Wu ◽  
Fergal Casey ◽  
Maureen Peterson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Locally Advanced ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Cancer Genes ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
The Impact ◽  
Generation Sequencing

9050 Background: Somatic mutations derived from the expansion of clonal populations of blood cells (clonal hematopoiesis of indeterminate potential, or CHIP) may be detected in sequencing of cell-free DNA (cfDNA) samples. We evaluated the potential implications of CHIP in targeted sequencing of lung cancer plasma samples using matched peripheral blood mononuclear cells (PBMC) to identify CHIP. Methods: Samples were evaluated from OAK, a phase 3 trial of atezolizumab in locally advanced or metastatic NSCLC following failure with platinum-based therapy. 94 samples from Cycle 1 Day 1 (C1D1) plasma and matched PBMC were analyzed with the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), a 198-kb next-generation sequencing panel targeting cancer genes. Plasma samples from subsequent cycles of therapy (C2D1, C3D1, and C4D1) were also sequenced with the same panel. Using median input amounts of 22.8 ng cfDNA and 50 ng PBMC DNA, we obtained median deduplicated depths of 5413 and 5070, respectively. Results: In C1D1 cfDNA, a median of 120 single nucleotide variants were detected per sample, with 5.13% of variants not identified in matched PBMC (i.e., putative tumor-derived somatic variants) versus 94.87% of variants identified in matched PBMC (i.e., germline or CHIP variants). While the majority of PBMC-matched variants were SNPs with allele frequency (AF) around 50% or 100% as expected, there was a median of 1 (range 0-8) PBMC-matched cfDNA variants per sample with AF below 10%. Consistent with CHIP, the number of PBMC-matched cfDNA variants per subject below AF 10% were positively associated with age (p-value = 0.0145), and TP53 was the most frequently mutated gene. We found similar results in plasma samples from subsequent cycles. Conclusions: Plasma and PBMC sequencing analysis identified potential mutations derived from CHIP. However, 39% of cfDNA samples had zero potential CHIP mutations identified in the study, possibly due to the specific regions targeted by the AVENIO assay. While this study suggests that only a small percentage of variants detected by the AVENIO Surveillance panel in lung cancer are derived from CHIP, further studies are warranted to assess the impact and removal of these variants.

scholarly journals Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss

2019 ◽  
Vol 10 ◽  
Author(s):  
Giorgia Girotto ◽  
Anna Morgan ◽  
Navaneethakrishnan Krishnamoorthy ◽  
Massimiliano Cocca ◽  
Marco Brumat ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Animal Models ◽  
Next Generation ◽  
Age Related ◽  
New Gene ◽  
Age Related Hearing Loss ◽  
Generation Sequencing

scholarly journals Recipients of Myelotoxic Chemotherapy Have Increased Prevalence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) with a Typical Distribution of Chip-Associated Mutations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3841-3841
Author(s):  
Adam J Olszewski ◽  
Anna Dorota Chorzalska ◽  
Annette S. Kim ◽  
Peter J. Quesenberry ◽  
Mary L Lopresti ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Cancer Survivors ◽  
Null Hypothesis ◽  
Research Funding ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
Myeloid Neoplasm ◽  
Typical Distribution ◽  
Generation Sequencing

Abstract Background: Recent studies (Coombs et al., Cell Stem Cell 2017) have identified presence of clonal hematopoiesis of indeterminate potential (CHIP) in samples of solid tumors. CHIP is more prevalent among cancer survivors who subsequently develop therapy-related myeloid neoplasm (Gillis et al., Lancet Oncol 2017; Takahashi et al., Lancet Oncol 2017; Gibson et al., J Clin Oncol 2017). However, the relationship between CHIP and exposure to myelotoxic chemotherapy delivered as part of treatment for solid tumor is uncertain. We hypothesized that CHIP is more prevalent among recipients of myelotoxic chemotherapy compared with age-matched population. Methods: In this prospective, cross-sectional study, we collected peripheral blood samples from survivors of breast cancer or lymphoma who had received anthracycline- and/or alkylator-containing chemotherapy as part of their curative cancer therapy. All subjects had to be clinically free of cancer, and not have any hematologic disorders or unexplained cytopenias. We recruited patients age 50 to 70, because according to published population datasets (Jaiswal et al. and Genovese et al., NEJM 2014) in a cohort with mean age of 60 the expected CHIP prevalence would be about 5%. To minimize any potential contamination by circulating tumor cells, we isolated genomic DNA from purified CD45+ cells. We determined presence of CHIP by next-generation sequencing using an Illumina TruSeq Custom Amplicon kit (MiSeq V2.2). The assay targeted 757 coding exons of 96 genes commonly mutated in hematologic malignancies, including 20 CHIP-defining genes. To establish presence of CHIP, we required a known pathogenic variant with variant allele fraction (VAF) ≥ 2%. According to a pre-specified statistical plan, assuming one-sided alpha of 0.05, the study had 80% power to reject the null hypothesis of baseline CHIP prevalence of 5% in a cohort with sample size of 80. Results: Among 80 enrolled subjects, median age was 62 years (interquartile range, 56-67). There were 78% women, and 88% of subjects were white non-Hispanic. Patients had received doxorubicin- and/or cyclophosphamide-containing adjuvant or curative chemotherapy for breast cancer (56%) or lymphoma (44%). Median time from completion of chemotherapy to enrollment was 27 months (interquartile range, 11-59). We have completed sequencing of 72 samples (updated analysis will be provided at the meeting). Mean coverage depth was 1418x (±224), and ≥200x coverage was achieved in a mean 91.4% (±1.8%) of target amplicons. We detected CHIP in 12 subjects (17%; binomial 95% confidence interval: 10-27%; P=.0002 for the null hypothesis test of 5% prevalence). Mean VAF for the CHIP mutations was 5.3% (range, 1.4% to 29.9%), and patients had up to 4 CHIP-associated mutations (Fig. A). The CHIP-associated mutations had a typical distribution with most common mutations in DNMT3A, ASXL1, SRSF2, and TET2 (Fig. B). There was only 1 TP53 mutation, previously suggested to associate with exposure to chemotherapy (Coombs et al., 2017). Potentially germline variants of unknown significance (VUS) were found in 78% of patients, at mean VAF 49% (Fig. C), most commonly in ATM (12%), NOTCH2 (7%), BCORL1, and DNMT3B (6% each). Additionally, 2 patients had low-VAF variants suspicious for CHIP: ATM c.6059G>A (3.0%); PRPF8 c.790T>C (VAF 2.3%). Presence of CHIP was not significantly associated with age (within the narrow age range in the study cohort), sex, race, type of cancer (breast or lymphoma), count of white cells, red cells, platelets, or time elapsed from completion of chemotherapy. Conclusions: We have detected a significantly increased, more than 3 times the expected value, prevalence of CHIP among cancer survivors who had received myelotoxic chemotherapy. However, the distribution of mutations was typical for CHIP, without previously suggested over-representation of TP53. Further research is ongoing to determine whether presence of CHIP is related to a direct mutagenic effect of chemotherapy or competitive advantage of pre-existing CHIP clones after the hematopoietic stress of chemotherapy. Our data indicate that an affordable next-generation sequencing screen may be useful for detection of CHIP in cancer patients who are planning adjuvant chemotherapy, or as a surveillance tool after such therapy, to predict the risk of a therapy-related myeloid neoplasm and optimize personalized treatment strategies. Disclosures Olszewski: TG Therapeutics: Research Funding; Genentech: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Kim:Aushon Biosciences: Consultancy; LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy. Fenton:Astellas Pharma US: Other: Spouse employment. Reagan:Alexion: Honoraria; Takeda Oncology: Research Funding; Pfizer: Research Funding.

scholarly journals The Clonal Hematopoietic Spectrum of Down Syndrome and ML-DS

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3839-3839
Author(s):  
R. Spencer Tong ◽  
Wing Hing Wong ◽  
David R. O'Leary ◽  
Kathleen S. Jay ◽  
D. Katherine Grange ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Next Generation Sequencing ◽  
Wnt Signaling ◽  
Error Rate ◽  
Conflicts Of Interest ◽  
Phase Iii ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
Recurrent Mutations ◽  
Generation Sequencing

Abstract Introduction: Children with Down syndrome (DS) have a 150-fold higher risk of developing myeloid leukemia (ML-DS) (Wechsler J, Nat Gen 2002). Ten percent (10%) of DS children are also predisposed to developing a preleukemic self-limiting condition, transient myeloproliferative disorder (TMD). While the majority of these children achieve complete remission, 20-30% of TMD patients will eventually develop ML-DS (Gamis AS, Blood 2011). Unique to ML-DS and TMD is the association with mutations in the X-linked hematopoietic transcription factor, GATA1 (Alford KA, Blood 2011), which are pathognomonic, but perhaps not sufficient, for ML-DS. Along with GATA1 mutations, ML-DS has a distinct mutational landscape implicating the cohesin complex, epigenetic regulators, signal transducers and the RAS pathway (Yoshida K, Nat Gen 2013). While these findings have informed our understanding of ML-DS etiology, ~30% of healthy DS neonates - without overt TMD or ML-DS - also harbor GATA1 mutations (Roberts I, Blood 2013), suggesting that ML-DS arises from clonal outgrowth of these progenitors that acquire specific additional mutations. Because the spectrum of physiologic clonal hematopoiesis is unknown in DS, we sought to characterize clonal mutations in healthy DS children as compared to a cohort of ML-DS cases using our highly sensitive and specific error-corrected sequencing (ECS) methodology. A precise understanding of their altered hematopoietic development may inform risk stratification, therapeutic selection and outcomes. Methods: The Druley lab has developed a custom error-corrected sequencing (ECS) panel targeting 80 genes frequently mutated in both pediatric and adult myeloid malignancies (Young AL, Nat Comm 2016) that is validated to identify clonal mutations as rare as 0.0001 variant allele fraction (VAF), 100X below the error-rate of next-generation sequencing (Wong TN, Nature 2015; Young AL, Leukemia 2015). Using ECS, we surveyed 102 DS children (47 ML-DS enrolled from a current Phase III ML-DS study; 55 healthy DS enrolled at St. Louis Children's Hospital). Results: We identified 294 total clonal variants in 72/102 children (60.0%, healthy DS; 83.0%, ML-DS cases) at 0.0002-0.82 VAF. On average, we found 1.8 clonal variants per healthy DS control and 4.1 clonal variants per ML-DS case. Consistent with Yoshida et al., we find a spectrum of recurrent mutations in ML-DS cases: GATA1 (53.2%), EZH2 (25.5%), RAD21 (14.9%), and STAG2 (10.6%) etc. Surprisingly, we also identified frequent mutations in FAT1 (14.9%) and SETD2 (10.6%), genes previously unassociated with ML-DS. Moreover, we report a different set of recurrent mutations in healthy DS children: FAT1 (12.7%), BCOR (12.7%), TET2 (10.9%), SETD2 (7.3%), and TRIM24 (5.5%), and TP53 (5.5%) etc. Discussion: Earlier ML-DS studies could only detect more common mutations >0.02 VAF due to the error-rate of standard next-generation sequencing (NGS). Using ECS, we present the first characterization of the unique clonal hematopoietic spectrum in children with DS and ML-DS. These results reveal that clonal hematopoiesis is common amongst healthy DS children without hematologic conditions, and that children with ML-DS have a higher clonal mutation burden than those without. Notably, we identified two novel genes recurrently mutated in both ML-DS cases and healthy DS controls: FAT1 and SETD2. We suspect that these mutations were not identified in other ML-DS studies as the median VAFs of our detected FAT1 and SETD2 mutations were 0.001 and 0.0008, respectively. Mutations in FAT1 and SETD2 have been shown to lead to dysregulated Wnt signaling in numerous cancers (Morris LG, Nat Gen 2013; Yuan H, J Clin Invest 2017). We and others have previously shown that Wnt signaling is crucial in specifying a primitive or definitive hematopoietic program (Sturgeon CM, Nat Biotech 2014; Creamer JP, Blood 2017). We speculate that these mutant FAT1 and SETD2 clones, like mutant GATA1 clones, may inhibit the definitive hematopoietic potential in DS children. Lastly, we note a reduced incidence of GATA1 mutations in our healthy DS cohort and attribute this to an older average age (8.3 years), as compared to Roberts et al. studying neonates. Other groups have speculated that somatic mutations in GATA1 (with a DS background) may only occur during a restricted developmental window (<4 years) as TMD clones cycle into quiescence afterwards (Zhe L, Nat Gen 2005; Hasle H, Leukemia 2007). Disclosures No relevant conflicts of interest to declare.

scholarly journals Quantitative and Qualitative QC of Next-Generation Sequencing for Detecting Somatic Variants: An Example of Detecting Clonal Hematopoiesis of Indeterminate Potential

2020 ◽  
Vol 66 (6) ◽  
pp. 832-841
Author(s):  
Young Kyu Min ◽  
Young Kee Lee ◽  
Seong-Hyeuk Nam ◽  
Jae Kyung Kim ◽  
Kyung Sun Park ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Reference Materials ◽  
Limit Of Detection ◽  
Read Depth ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Clonal Hematopoiesis ◽  
Fold Reduction ◽  
The Cost ◽  
Generation Sequencing

Abstract Background Because next-generation sequencing (NGS) for detecting somatic mutations has been adopted in clinical fields, both qualitative and quantitative QC of the somatic variants through whole coding regions detected by NGS is crucial. However, specific applications or guidelines, especially for quantitative QC, are currently insufficient. Our goal was to devise a practical approach for both quantitative and qualitative QC using an example of detecting clonal hematopoiesis of indeterminate potential (CHIP). Methods We applied the QC scheme using commercial reference materials and in-house QC materials (IQCM) composed of haplotype map and cancer cell lines for monitoring CHIP. Results This approach efficiently validated a customized CHIP NGS assay. Accuracy, analytical sensitivity, analytical specificity, qualitative precision (concordance), and limit of detection achieved were 99.87%, 98.53%, 100.00%, 100.00%, and 1.00%, respectively. The quantitative precision analysis also had a higher CV percentage at a lower alternative read depth (R2 = 0.749∼0.858). Use of IQCM ensured more than 100-fold reduction in the cost per run compared with that achieved using commercial reference materials. Conclusion Our approach determined the general analytical performance of NGS for detecting CHIP and recognized limitations such as lower precision at a lower level of variant burden. This approach could also be theoretically expanded to a general NGS assay for detecting somatic variants. Considering the reliable NGS results and cost-effectiveness, we propose the use of IQCM for QC of NGS assays at clinical laboratories.

Identification of clonal hematopoiesis mutations in solid tumor patients undergoing unpaired commercial next-generation sequencing assays.

2018 ◽  
Vol 36 (15_suppl) ◽  
pp. 12068-12068
Author(s):  
Catherine Callaghan Coombs ◽  
Nancy Gillis ◽  
Kelly L Bolton ◽  
Jonathan S. Berg ◽  
Xianming Tan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumor ◽  
Next Generation ◽  
Tumor Patients ◽  
Clonal Hematopoiesis ◽  
Generation Sequencing

scholarly journals Next-generation sequencing identified SPATC1L as a possible candidate gene for both early-onset and age-related hearing loss

2018 ◽  
Vol 27 (1) ◽  
pp. 70-79 ◽  
Author(s):  
Anna Morgan ◽  
Dragana Vuckovic ◽  
Navaneethakrishnan Krishnamoorthy ◽  
Elisa Rubinato ◽  
Umberto Ambrosetti ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Candidate Gene ◽  
Early Onset ◽  
Next Generation ◽  
Age Related ◽  
Age Related Hearing Loss ◽  
Generation Sequencing

scholarly journals Identification of Clonal Hematopoiesis Mutations in Solid Tumor Patients Undergoing Unpaired Next-Generation Sequencing Assays

2018 ◽  
Vol 24 (23) ◽  
pp. 5918-5924 ◽  
Author(s):  
Catherine C. Coombs ◽  
Nancy K. Gillis ◽  
Xianming Tan ◽  
Jonathan S. Berg ◽  
Markus Ball ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumor ◽  
Next Generation ◽  
Tumor Patients ◽  
Clonal Hematopoiesis ◽  
Generation Sequencing
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