Background The increasing use of next-generation sequencing (NGS) has identified a subset of patients who harbor somatic mutations associated with myeloid malignancies despite having no overt hematologic cancer—a condition termed clonal hematopoiesis (CH). Due to the invasiveness of bone marrow aspiration, peripheral blood NGS is increasingly used to screen for possible myeloid neoplasms. Identified mutations are stratified into 3–4 tiers based on their clinical significance using established databases. However, the relationship between mutation tier and hematological markers or comorbidities is not well characterized. We conducted a single-center retrospective cohort study to evaluate the severity of hematologic abnormalities, associated clinical factors, and progression to myeloid malignancy in patients without a diagnosed myeloid neoplasm, stratified by CH mutation tier.

Methods NGS assays were performed using NYU-Myeloseq, a 50-gene panel targeting hematologic malignancies, on peripheral blood samples collected at NYU Langone between January 2021 and December 2023. Patients diagnosed with myeloid malignancy within 4 weeks of NGS testing were excluded. Patients were categorized into two groups based on mutation tier: CH+ (tier 1 or 2 mutations) and CH- (tier 3 mutations, including variants of unknown significance [VUS]). All patients underwent NGS for benign hematologic conditions, classified into five categories: cytopenia, cytosis (leukocytosis, polycythemia, thrombocytosis), mixed cytosis/cytopenia, presence of immature granulocytes, or splenomegaly/other.

Statistical analyses included Chi-square, Fisher's exact, and Kruskal-Wallis tests to assess differences in complete blood count (CBC) with differential, comorbidities, demographics, smoking status, BMI, and ECOG performance status.

Results Among 1,749 patients who underwent NGS, 969 had at least one somatic mutation. Of these, 195 (19.2%) were in the CH+ group and 774 (78.2%) in the CH- group. The CH+ group was significantly older (mean age 74 vs. 55 years, p<0.001). Tier 1/2 mutations were more prevalent in males than females (26% vs. 15%, p<0.001) and in smokers compared to non-smokers (28% vs. 17%, p=0.001). Poor functional status (ECOG ≥ 2) was also more common in the CH+ group (10.3% vs. 4.5%, p=0.001).

CH+ mutations were associated with higher prevalence of comorbidities: COPD (29% vs. 21%, p=0.018), diabetes (30% vs. 21%, p=0.006), renal failure (18% vs. 9.9%, p=0.002), and solid tumors (27% vs. 14%, p<0.001).

There were 37 distinct gene mutations observed. In the CH+ group, 65% had a single mutation and 37% had ≥2; the average variant allele frequency (VAF) was 43%. In contrast, 64% of the CH- group had ≥2 mutations with an average VAF of 49%.

In patients tested for cytopenia, the CH+ group had more pronounced hematologic abnormalities: lower hematocrit (33.9 vs. 36.6, p=0.004), lower hemoglobin (11.0 vs. 11.9, p=0.01), higher MCV (96 vs. 93, p<0.001), and higher WBC count (5.6 vs. 4.5, p=0.016). Among this subgroup, CH+ mutations were again more common in smokers (58.6% vs. 38.8%, p=0.009) and patients with solid tumors (33% vs. 22%, p=0.048).

A total of 97 patients underwent bone marrow biopsy at least four weeks after peripheral blood NGS—44 from the CH+ group and 53 from the CH- group. Myeloid malignancy was confirmed in 57% of CH+ patients versus 11% of CH- patients (p<0.001). Among those who developed myeloid malignancy, 55% had ≥3 mutations, compared to 23% in those without malignancy (p=0.03).

Conclusions Peripheral blood NGS is a valuable screening tool for evaluating patients with unexplained cytopenias or cytoses. Detection of tier 1 or 2 mutations (CH+) significantly correlated with a higher likelihood of bone marrow-confirmed myeloid malignancy (57% of cases). A greater number of distinct mutational variants detected was associated with an increased likelihood of developing a myeloid neoplasm. CH+ status was also associated with older age, smoking history, poor functional status, and specific comorbidities such as COPD, diabetes, renal failure, and solid tumors. Hematologic abnormalities (e.g., lower hgb/hct, higher MCV and WBC) were more pronounced in CH+ patients. Notably, 11% of patients with only tier 3 mutations also developed myeloid malignancy, suggesting that some VUS may carry pathogenic potential. These findings support the utility of peripheral blood NGS and highlight the need for further prospective studies with long-term follow-up.

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2025
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