Prevalence of Clonal Hematopoiesis and Mutation Patterns in Unexplained Anemia of Community-Dwelling Elderly Individuals: A Case-Control Study

The prevalence of anemia in the general population rises sharply after the age of 50, reaching 20% or more in individuals aged 85 years or older. In up to one third of cases, the origin of anemia remains unexplained, i.e. not attributable to causes detectable with conventional tests or to any concomitant diseases, and several potential mechanisms have been proposed, including myelodysplasia (MDS) (PMID 15238427). Recent studies showed that somatic mutations are commonly acquired in hematopoietic cells during aging, and are associated with increased risk of hematologic malignancies (PMID 25426837; 25426838). In a recent analysis in patients with unexplained cytopenia undergoing a comprehensive diagnostic workup, we found that mutation profiling was highly predictive of myeloid neoplasm (PMID 28424163).

In this study, we performed a mutation analysis in a cohort of elderly individuals with unexplained anemia and in sex- and age-matched control subjects both selected from a population-based study, as well as in a matched group of patients with MDS, with the aim to investigate the relationship between clonal hematopoiesis, mutation profile and unexplained anemia in community-dwelling elderly subjects.

Seventy individuals aged 60 years or older having anemia according to WHO criteria, not explained by nutritional deficiency, renal failure or inflammation, and 105 sex- and age-matched control subjects (ratio 1:1.5) were selected from the "Val Borbera" Study, a population-based study recruiting a total of 1803 people. In addition, 105 matched control patients with newly diagnosed MDS without excess blasts was analyzed. Median age was 78 years (range 62-102), 133 individuals were males, 147 females. A panel of 54 genes was analyzed on peripheral blood cells, using a HiSeq 2500 platform (Illumina, San Diego, CA) (median depth 1550, range 845-2057).

Among community-dwelling individuals, a significant effect of age was observed on mutation prevalence (P=.023); however, this effect was limited to controls. A significantly higher mutation rate was observed in cases compared with controls (42.9% vs 24.8%, P=.014). This rate was even higher when restricting the analysis to individuals with hemoglobin <12.5 g/dL in males and <11.5 g/dL in females and/or MCV>105 fL (63% vs 24.8%, P<.001). A significantly higher Variant Allele Frequency (VAF) was found in cases compared with controls (median value 0.21, range 0.02-0.51 vs 0.07, 0.02-0.38; P=.035). A significantly higher number of individuals with two somatic mutations was found among cases compared with non-anemic controls (14.3% vs 1.9%, P=.003). Subjects with two somatic mutations showed a significantly higher VAF compared with those having an isolated mutation (median value 0.31, range 0.03-0.50 vs 0.06, 0.06-0.51; P=.008). In a multivariable regression analysis, number of mutations per subject and VAF were independent predictors of anemia (OR=7.55, P=.03, and OR=1.67, P=.037, respectively). A significantly higher prevalence of splicing factor mutations was found in cases compared with controls (11.1% vs 0%, P=.008). In most individuals with multiple mutated genes, mutation pattern included a DNA methylation gene mutation associated with a subclonal mutation.

When comparing community dwelling individuals with unexplained anemia with a matched group of patients with MDS, a significantly higher number of subjects with two or more mutations was found in MDS compared with unexplained anemia (47.6% vs 14.3%, P<.001). MDS patients showed a significantly higher VAF compared with individuals with unexplained anemia (median value 0.41, range 0.02-0.93 vs 0.21, 0.02-0.51; P<.001). Finally, a significantly higher prevalence of splicing factor mutations was observed in MDS compared with unexplained anemia (55.2% vs 11.1%, P<.001).

In conclusion, this study provides evidence of a high rate of clonal hematopoiesis in community-dwelling elderly individuals with unexplained anemia compared with matched controls with normal hemoglobin values, mainly sustained by an enrichment in subjects with accumulation of mutations proven highly predictive of myeloid neoplasm with myelodysplasia. Mutation analysis on peripheral blood cells may complement current diagnostic approach to unexplained anemia in the frail population of elderly individuals, increasing compliance to diagnostic tests and overall the diagnostic accuracy of myeloid neoplasms.