The use of targeted sequencing and flow cytometry to identify patients with a clinically significant monocytosis

Abstract

The diagnosis of chronic myelomonocytic leukemia (CMML) remains centered on morphology, meaning that the distinction from a reactive monocytosis is challenging. Mutational analysis and immunophenotyping have been proposed as potential tools for diagnosis; however, they have not been formally assessed in combination. We aimed to investigate the clinical utility of these technologies by performing targeted sequencing, in parallel with current gold standard techniques, on consecutive samples referred for investigation of monocytosis over a 2-year period (N = 283). Results were correlated with the morphological diagnosis and objective outcome measures, including overall survival (OS) and longitudinal blood counts. Somatic mutations were detected in 79% of patients, being invariably identified in those with a confirmed diagnosis (99%) but also in 57% of patients with nondiagnostic bone marrow features. The OS in nondiagnostic mutated patients was indistinguishable from those with CMML (P = .118) and significantly worse than in unmutated patients (P = .0002). On multivariate analysis, age, ASXL1, CBL, DNMT3A, NRAS, and RUNX1 mutations retained significance. Furthermore, the presence of a mutation was associated with a progressive decrease in hemoglobin/platelet levels and increasing monocyte counts compared with mutation-negative patients. Of note, the immunophenotypic features of nondiagnostic mutated patients were comparable to CMML patients, and the presence of aberrant CD56 was highly specific for detecting a mutation. Overall, somatic mutations are detected at high frequency in patients referred with a monocytosis, irrespective of diagnosis. In those without a World Health Organization–defined diagnosis, the mutation spectrum, immunophenotypic features, and OS are indistinguishable from CMML patients, and these patients should be managed as such.

Oligomonocytic and overt chronic myelomonocytic leukemia show similar clinical, genomic, and immunophenotypic features

“…Interestingly, patients with OM-CMML who had TET2 mutations had MO1 percentage .94% in a rate similar to those with overt CMML. Moreover, as previously reported by Cargo et al, 31 CD56 positivity in monocytes was significantly associated with TET2 mutation in our series. These findings suggest that the impairment of this pathway could be the pathophysiological hallmark of these entities.…”

“…This mutation was the only one of the assessed mutations that enabled division of the OM-CMML series into 2 groups, which showed a significant difference in the proportion of patients with MO1 percentage .94% (supplemental Table 3). As published by Cargo et al, 31 CD56 positivity in monocytes correlated positively with TET2 mutation in our series, both as a binary value (F coefficient, 0.45; P , .001) or as a continuous variable (r Spearman, 0.4; P , .001). Likewise, the median expression of CD56 in monocytes was significantly higher in the patients with TET2 mutations (34% vs 3%; P , .001), and the proportion of patients showing CD56 positivity was also higher in the TET2-mutated group (75% vs 24%; P , .001).…”

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma

“…Again, this variant was represented among a cohort of patients with myeloid malignancy (2/1221 patients with AML, 2/286 patients with chronic myelomonocytic leukemia; supplemental Table 4). 32 Immunoblotting of PBMCs confirmed loss of TET2 protein expression in the proband, whereas heterozygous relatives showed intermediate levels relative to wt control (Figure 2C).…”

“…Furthermore, we identified the same variant in heterozygosity in patients with myeloid malignancy (3/1221 patients with acute myeloid leukemia [AML] and 1/286 patients with chronic myelomonocytic leukemia; supplemental Table 4). 32 The expression of TET2 H1382R protein was not impaired relative to TET2 wt in primary cells (Figure 2C) or in a recombinant system (Figure 2D). We compared the enzymatic activity of TET2 wt and TET2 H1382R by immunofluorescence microscopy analysis of transfected HEK293T cells stained for 5hmC.…”

Diagnosis and treatment of chronic myelomonocytic leukemia

“…The co-occurrence of monocytosis and these molecular features are important in distinguishing reactive monocytosis from clonal diseases such as CMML. When the target sequencing results of 283 patients with monocytosis were previously analyzed, ≥1 gene mutations were found in 57% of 65 patients with monocytosis but without myeloid disease in the bone marrow diagnosed according to the WHO classification [33]. The most common gene mutations found in these patients were in TET2, SRSF2, and ASXL1, as seen in CMML patients.…”