Mutational Profiling of Peripheral Blood and Bone Marrow Samples Discriminates Reactive Monocytosis from Chronic Myelomonocytic Leukaemia

Background

Chronic myelomonocytic leukaemia (CMML) presents a diagnostic challenge to the haematologist. Distinguishing between a reactive monocytosis and clonal expansion is difficult, and current diagnostic criteria allow for a diagnosis of CMML even in the absence of a clonal marker of disease as long as the monocytosis is persistent. This fails to correctly identify patients with prolonged reactive changes, increasing mis-diagnoses. More recently, large sequencing studies have identified somatic mutations in >90% of patients with CMML, providing potential objective evidence to support a diagnosis. To investigate the utility of high throughput sequencing to discriminate clonal disease and therefore improve diagnosis of CMML, we performed mutational analysis on all samples referred for investigation of a monocytosis to the Haematological Malignancy Diagnostic Service (HMDS) over a 2 year period. The aim of this study was to determine the frequency of mutations in this patient group and whether the presence of mutations can predict disease and outcome.

Methods

Samples from all patients (initial and follow-up) referred to HMDS with a monocytosis or suspected CMML between July 2014-July 2016 were included in the study. Those with a previous history of a myeloid malignancy diagnosed before July 2014 were excluded. 377 samples from 297 patients were processed and reported according to using current gold standard techniques, with targeted sequencing of 27 recurrently mutated genes in myeloid malignancies performed in parallel. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Detected variants were reported down to a minimum variant allele fraction of 5% and coverage of 100X. Low level variants were confirmed by repeat sequencing and SRSF2 regions were infilled using Sanger sequencing. Data from the literature as well as public online databases (dbSNP, COSMIC, ClinVar) and Alamut Visual were evaluated to annotate likely pathogenic variants.

Results

Of the164 patients who presented with an initial bone marrow sample, 95 had a confirmed diagnosis of CMML, of which 93 had a demonstrable mutation (98%). The spectrum of mutations in this group reflects that reported in the literature with TET2, SRSF2 and ASXL1 being most frequently mutated. A further 15 patients, all with mutations, were diagnosed with an alternative myeloid malignancy.

In those without a confirmed diagnosis by conventional means, a somatic mutation was detected in 62% (39/54) of cases. Importantly, those with a mutation had both phenotypic and genotypic features indistinguishable from the CMML group. In particular CD56 overexpression by immunophenotyping was found almost exclusively in patients with a mutation whether a diagnosis was confirmed or not. To date, a follow-up sample has been received from 7 mutation-positive patients, of which 5 were diagnostic. All 5 cases had identical mutational profiles in the paired samples.

In 133 patients, a peripheral blood sample was received as the initial specimen and 71% (94/133) of these harboured a mutation. As yet, 94 patients have not had a subsequent bone marrow biopsy so a conventional diagnosis has not been made.

In those with a follow-up bone marrow sample (n=39), the mutational profile between paired samples was found to be highly concordant (98%). In addition, the detection of a mutation in the peripheral blood was strongly predictive of a myeloid malignancy in the bone marrow. Of the 30 patients with a detectable mutation, 29 had a confirmed diagnosis, including 20 patients with CMML, 5 with AML and 4 with other chronic myeloid malignancies. The remaining 9 patients without a mutation showed no morphological evidence of disease in the bone marrow (sensitivity 96.7%; CI 82.8%-99.9%, specificity 100%; CI 66.4%-100%).

Conclusion

This study has shown that patients investigated for a monocytosis commonly harbour somatic mutations which can be detected in the peripheral blood at a high frequency and with high confidence. The presence of a mutation correlates strongly with a CMML phenotype and detection in the peripheral blood is strongly predictive of a bone marrow diagnosis. Screening of peripheral blood samples using a targeted gene panel provides a highly effective tool to diagnose CMML. Follow-up of this patient group is ongoing and updated results will be available for presentation at the meeting.