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Clonal hematopoiesis and AML risk
from 20 adult women without AML revealed that nearly all studied individuals harbored somatic mutations fre- quently observed in myeloid malignancies. The detected hematopoietic clones were often stable over the ten years between blood collections and did not demonstrate posi- tive selection or clonal expansion, regardless of the gene mutated. It is important to note that clonal mutations at a lower frequency than 0.02 VAF are currently not regarded as CHIP, and their clinical significance is even less well understood. The two aforementioned studies of pre-diag- nosis CHIP observed associations of increased AML risk for persons with clones ≥0.005 or ≥0.01 VAF over a shorter follow-up period.4,5 The present investigation examined whether detection of lower-VAF clones or specific muta- tions are associated with future risk of AML in a nested case-control sample (35 cases, 70 controls) from the Nurses’ Health Study (NHS) and Health Professionals Follow-up Study (HPFS) cohorts with up to 22 years of fol- low up after sample collection.8,9 We also investigated whether clonal evolution over ten years was associated with long-term future risk of AML in 11 women in the NHS with multiple pre-diagnosis samples.
Methods
Study population
Details of the NHS and HPFS design and data collection and fol- low-up methods are published elsewhere8,9 (see also Online Supplementary Methods). Biennial questionnaire return rates have been consistently high (>95% in the blood subcohorts described below).
Blood subcohorts
The “blood subcohorts” comprise 32,826 women (NHS) who provided a heparinized whole blood sample from 1989-1990,10 of whom 18,743 provided a second whole blood sample from 2000- 2001,11 as well as 18,018 men (HPFS) who provided an EDTA whole blood sample from 1993-1995. Participants provided writ- ten informed consent. The present study protocol was approved by the Institutional Review Boards of Brigham and Women’s Hospital, Harvard TH Chan School of Public Health and Washington University.
Case and control selection
The present study utilized a nested case-control design for which the case definition included all blood subcohort participants with confirmed diagnoses of AML (ICD-8=205.0) occurring after blood draw. We matched two controls per case on cohort (sex), race, birthdate (±1 year), and blood draw details (date ±1 year, time ±4 hours, fasting status). For NHS cases with a second collec- tion sample, we matched controls with a second sample using the same criteria. These protocols selected 35 cases (16 NHS, 19 HPFS) and 70 controls (32 NHS, 38 HPFS), including 11 matched sets (NHS) with two samples (n=137 total samples after excluding four with insufficient volume).
Clonal hematopoiesis of indeterminate potential determination and validation
Sequencing libraries were prepared as previously described6 using the Illumina TruSight Myeloid Sequencing Panel for targeted capture from 54 leukemia-associated genes (Online Supplementary Methods and Online Supplementary Table S1). Libraries were sequenced on the Illumina HiSeq 3000 platform per manufacturer specifications; with technical replicate libraries sequenced on dif-
ferent machine runs. ECS analysis of raw sequencing results was performed as previously described,6 except that, to improve rare SNV identification at potential “hot spot” loci, we re-called vari- ants from the binomial error model after removing the variants already identified until a subsequent iteration revealed no addi- tional new variants. We reported single nucleotide variants (SNV) and insertions and deletions (indels) identified in both technical replicates for a given sample. To validate our ECS-based variant calls, we performed droplet digital polymerase chain reaction (ddPCR) for 61 variants.
Statistical analysis
We combined NHS and HPFS data to maximize statistical power. We analyzed mutations detected in both technical repli- cates for ≥4 participants and selected VAF thresholds (≥0.001, ≥0.005, ≥0.01, ≥0.02), in the first collection samples and in samples from either collection. We used conditional logistic regression, conditioning on matched sets, to calculate odds ratios (OR) and 95% confidence intervals (CI) for the relative risk of AML for a given detected variant or VAF threshold. Sparse data precluded evaluation of confounding by other AML risk factors3,12 or effect modification. Exploratory and sensitivity analyses are detailed in the Online Supplementary Methods. We utilized SAS version 9.3 for statistical analyses and the ggplot2 and ppcor packages of R ver- sion 3.3.313 for graphical descriptive analyses. Hypothesis tests assumed a two-tailed a-error of 0.05.
Results
Study samples
Due to the matched design, the cases and controls had similar distributions of sex, age at blood collection, and interval from blood draw to case diagnosis or control index date (Table 1). The median age of sample collection was 61 years for the first collection and 70 years for the second collection. The median age of AML diagnosis was 76 years (range: 53-87 years). More than 90% of cases and controls had one year or more of follow up after blood draw, and >88% of each group had follow-up intervals of five or more years. All the women with repeat blood sam- ples had at least one year of follow up after the second blood collection (Table 1). All the participants selected into the study sample had self-reported their race/ethnicity as White.
Error-corrected sequencing results
During ECS library preparation, we generated an aver- age of 60 million raw sequenced reads, yielding 3.9 million ECS reads, per library, which translated into approximate- ly 8,000x ECS read coverage of the target space. We iden- tified 563 single nucleotide variants and 35 insertion/dele- tion (indel) variants by ECS; this corresponded to detec- tion of AML-associated mutations in 97% of all partici- pants (598 mutations, 5.8/person), with an average of 7.4 (range: 1-14) per case and an average of 5.0 (range: 0-15) per control (Online Supplementary Table S2). As expected, due to the targeted enrichment sequencing scheme, these mutations predominantly occurred in exonic regions (Online Supplementary Figure S1A). Most detected muta- tions were predicted to change the underlying amino acid sequence in cases and controls (Online Supplementary Figure S1B). Of the 252 clonal mutations detected in the cases, we identified 144 non-synonymous SNV (57%), 40 stop gain variants (16%), 22 intronic variants (9%), 18 indels
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