Somatic Mutations in TET2, but Not SF3B1, Are Prevalent during Normal Aging Hematopoiesis in Human

Abstract BACKGROUND. Somatic mutations acquired in the course of a lifetime contribute to the aging process and to the development of age-associated diseases including cancers. Our capacity to identify such events in the normal aging population prior to the appearance of malignancy is of utmost clinical importance to devise prevention or early intervention strategies. This is particularly relevant to myeloid cancers such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) which incidence increases dramatically with age. Recently, next-generation sequencing (NGS) efforts have helped to establish lists of recurrently mutated genes in these cancers. With these lists of candidate genes in hand, it is now possible to look for the presence of low frequency pre-malignant somatic lesions in the aging population. The contribution of these mutations to cancer development can then be assessed in longitudinal studies. We have previously shown (Nature Genet, 2012; 44:179), that somatic mutations in the epigenetic regulator gene TET2 occur in the normal aging population and is associated with clonal derivation of cells. However, little is known about the occurrence of mutation in other categories of frequently mutated gene in MDS such as the splicing factor SF3B1, which is the second mostly frequently mutated gene after TET2. SUBJECTS AND METHODS. We have selected 217 individuals based on age (>70 years) and clonality status from a well characterized cohort of normal aging individuals (n=4000). This cohort is comprised of women mostly aged greater than 60 years and without any known hematological disorder (medical history reviewed and normal complete blood counts at enrollment). We obtained blood cells and buccal epithelial cells from each subject. Blood cells were separated into polymorphonuclear (PMN) cells and mononuclear cells using standard procedures. T cells (CD3+) were further isolated from mononuclear cells. DNA and RNA were then isolated from all cell populations. X-chromosome inactivation (XCI) patterns at the HUMARA locus was determined in PMN, T-cells to assess the clonality of myeloid derived cells. MUTATIONAL ANALYSIS. We have developed a custom next generation sequencing approach using Ion AmpliSeq libraries on an Ion Torrent PGM sequencer. Extensive experimental validation and algorithm optimisation were performed to ensure the quality of the method, control experiments with positive and negative controls were done to demonstrate the specificity, while dilution curves were run to validate the sensitivity of mutation detection down to 5% variant allele frequency (VAF) at 500x mean coverage. The final validated design is composed of 65 amplicons spanning 9.78kb covering TET2 at 100% and 53 amplicons spanning 8.84kb covering SF3B1at 95.61% including all known hot spots. RESULTS. 14/207 subject had acquired (present in PMN, not in T-cells or epithelial cells) mutation in the TET2 gene in line with our previous results. The VAF varied between 6 and 47%, all mutation were further validated by Sanger sequencing. In contrast to TET2, no subject had mutation in SF3B1gene. CONCLUSION. Mutation in the SF3B1gene does not occur at a significant frequency in the aging population suggesting that alteration of this gene is not an early initiating event in the pathogenesis of MDS. This further suggest that in contrast to the extended number of genes mutated in MDS, only a limited number are likely to be found and implicated in the pre-leukemic phase. It is possible that epigenetic alterations, and not dysfunction of the spliceosome, could be a driving force underlying the pathogenesis of MDS. Complete exome sequencing and iterative studies will help decipher the sequence of events leading to age-associated myeloid cancer. Disclosures No relevant conflicts of interest to declare.

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Intrapatient Functional Clonality Deconvoluted By Coupling Intracellular Flow Cytometry and Next Generation Sequencing in Human Leukemia

Blood ◽

10.1182/blood.v128.22.3161.3161 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3161-3161

Author(s):

Qing Zhang ◽

Markus Ball ◽

Zhao Yulong ◽

Maria E. Balasis ◽

Alan F List ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Somatic Mutations ◽

Mononuclear Cells ◽

Leukemic Cells ◽

Human Leukemia ◽

Next Generation ◽

Roc Curve Analysis ◽

Functional Differences ◽

Generation Sequencing

Abstract Leukemic clones within the same patient have unique sensitivities to drugs in vivo and may have unique capacities to engraft within xenograft models suggesting that there exist intra-patient functional clonal differences. However, methods to detect the functional differences assigned to genetically defined clones remain limited. Here, we describe a method to measure functional differences of clones within the same leukemia patient by coupling intracellular flow cytometry and next generation sequencing (NGS). We hypothesize that functionally distinct clones can be inferred by stimulating cells with cytokines, fluorescently staining with a relevant downstream marker of activation, and then isolating functionally distinct fractions by flow sorting. NGS can then digitally annotate somatic variants in sorted, functionally distinct samples that can be compared to the bulk leukemic cell population. To achieve this, we optimized a DNA isolation method that results in high-quality DNA despite originating from fixed/permeabilized cells. In this way, we annotate enrichment and de-enrichment of variants upon stimulation and infer the functional clonality of any given sample. To provide proof-of-concept, we explored the well-established relationship between Chronic Myelmonocytic Leukemia (CMML) and GMCSF. CMML cells are uniquely hypersensitive to GMCSF and simultaneously have a well-established mutational composition that can be annotated by sequencing only 49 genes. This allowed us to leverage an amplicon-based enrichment panel that employs emulsion PCR to achieve reproducible library preparation with 5 ng of input DNA hence bypassing the need for whole genome amplification. We tested our methodology by mixing two cell lines whose response to GMCSF was known and somatic variants had been annotated a priori. K562 (non-GMCSF responsive) and THP-1 (GMCSF responsive) leukemic cells were mixed at 1:1 and then stimulated with GMCSF. The resultant cells were stained with anti-pSTAT5 and cell sorting isolated positive and negative fractions. Unstimulated mixed cells and each positive and negative fraction was sequenced and variant allele fractions (VAF) was compared to unstimulated bulk (Figure 1A). To quantitate the change in VAF, we reasoned that summing the difference in VAF of unsorted/pSTAT5+ and unsorted/pSTAT5- populations would yield the most informative composite value as it would contain information about VAF difference for each mutation in both the GMCSF stimulated and GMCSF unstimulated fractions. Using this method, we found that 100% of THP1 and K562 variants were enriched in the pSTAT5+ and pSTAT5- fractions, respectively (Figure 1B). Receiver Operator Curves (ROC) demonstrated that the data functionally discriminated mixed THP-1 and K-562 cells with an AUC of 0.9868 (p<0.0001) (Figure1C). We additionally performed serial dilutions of THP-1 to K562 cell mixtures and demonstrated cell number dependent recovery of THP-1 variants upon stimulation with GMCSF. Next we reasoned that somatic mutations should be uniquely augmented by GMCSF stimulation making it possible for somatic variant identification. To test this, we sequenced 8 CMML bone marrow mononuclear cells evaluated in the same way as above. We additionally sorted CD3+ cells as germline controls to identify somatic mutations. Using our 49-gene panel, we identified a total of 35 somatic variants and 160 germline variants. ROC curve analysis confirmed that our assay was capable of discriminating germline from somatic mutations with an AUC of 0.9388 (p<0.001). In a representative CMML case, we counterstained with CD33 to isolate myeloid progenitors and inferred a functionally distinct clone that was hypersensitive to GMCSF and uniquely carried a JAK2 and PTPN11 mutation that was obscured by bulk leukemia sequence (Figure 2). This inference was orthogonally validated by sequencing individual colonies from the same case grown under GMCSF conditions (Figure 3). Further validation is ongoing comparing mutations identified at engraftment of cells differentially xenografted in NSG and NSGS mice that differ only in their production of human cytokines. Taken together, we describe a novel method to annotate functional differences among mutationally-defined populations and identify functionally distinct clones in primary leukemia samples. Disclosures Komrokji: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

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