To the Editor
Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous myeloid neoplasm
that combines myelodysplastic and myeloproliferative features, and carries a poor
prognosis due to progression to acute myeloid leukemia or complications of cytopenias.
TET2, SRSF2 and ASXL1 are the most commonly mutated genes in CMML, but somatic variants
in additional genes have been identified
1–3
. Allogeneic stem cell transplantation is potentially curative, but most patients
are ineligible due to advanced age and/or co-morbidities. Hypomethylating agents (HMAs)
such as 5-azacitidine (5-Aza) induce responses in ~40% of CMML patients, but their
impact on survival remains debatable
4
. While TET2 mutations have been reported to predict HMA response in myelodysplastic
syndromes, data from patients treated with decitabine suggest that epigenetic profiles
rather than somatic mutations govern response to HMAs in CMML
5, 6
. Previous work described unchanged mutant allele burden in CMML in patients responding
to HMAs
1
, but detailed analyses of clonal evolution in relation to HMA response have not been
reported. We applied SubcloneSeeker computational analysis algorithm to whole exome
sequencing (WES) and single nucleotide polymorphism (SNP) array data to uncover clonal
architecture and evolution in CMML patients treated with 5-Aza on a prospective trial
7
. Compared to targeted sequencing, this approach allows reconstruction of clonal architecture
using all somatic mutation calls, including driver and passenger mutations, in an
unbiased fashion.
We studied a total of 46 samples obtained from twelve patients treated with single-agent
5-Aza for up to two years. All patients provided informed consent for the study. Ten
patients were treated on a prospective clinical study (NCT01350947) and two in analogy
to the study protocol. Median age at presentation was 70 years. Ten patients had CMML-1
and two had CMML-2. Three patients (25%) achieved complete remission (CR) and four
(33%) partial remission (PR), one had stable disease (SD), and one had no response
(International Working Group 2006 response criteria). Three patients (25%) progressed
after a period of SD (Supplementary Table 1).
Mononuclear cells (MNCs) or monocytes were isolated from bone marrow or blood samples
prior to treatment, at 3- and 6-month intervals on therapy, and at the end of study
or disease progression. Cultured mesenchymal stromal cells (n=4), skin fibroblasts
(n=6) or fluorescence-activated cell-sorted CD3+ lymphocytes (n=2) were used as sources
of constitutional DNA in CMML patients. WES was performed on paired tumor and control
samples, with median of 3 longitudinal tumor samples per patient (n=46). Copy number
variation (CNV) and loss of heterozygosity (LOH) were analyzed by whole-genome SNP
arrays (Infinium Omni2.5-8 v1.3, Illumina). Variant allele frequencies (VAFs) were
corrected for proportion of lymphocytes in MNC samples.
A median of 39 (range: 10 – 95) somatic mutations per patient exome was detected,
with average read depth (DP) of >200X per sample. The most common variants were non-synonymous
missense single-nucleotide variants (SNVs) (90%), followed by frameshift insertions/deletions
and stop-gain mutations (Supplementary Figures 1a-c). The variants were predominantly
transitions (65%) with transition-transversion ratio of 1.86, similar to other myeloid
and lymphoid malignancies
8, 9
. Mutations in TET2 and SRSF2 were each found in 62% of the patients. Additional mutations
detected in genes associated with CMML included ASXL1 (38%), RUNX1 (38%), CBL (31%),
KRAS (23%), DNMT3A (15%) and NRAS (15%) (Figure 1a). TET2, SRSF2 and ASXL1 mutations
persisted across pre- and post-treatment samples (Figure 1a). Two patients showed
copy neutral (CN)-LOH. CNV and mutations with corresponding VAFs in longitudinal samples
are summarized in Supplementary Table 2. Our data revealed higher median somatic mutations
per exome than a previous report
1
, probably related to higher average read depth. However, somatic mutation burden
was remarkably stable despite response to 5-Aza. Of 477 total somatic mutations identified
prior to 5-Aza, 98% were still detectable in the last follow-up samples and only 13
new variants were acquired on therapy.
To map clonal architecture, we used the SubcloneSeeker computational algorithm to
construct a set of clonal trees by clustering all somatic variants with similar VAFs
and calculating their cellular prevalence values
10
. Compatible trees from multiple longitudinal samples at different time points were
merged to establish a unified model of clonal evolution in each patient (Supplementary
Methods). Clonal evolution patterns were studied in patients without LOH, based on
changes in relative proportions of parental clones, pre- and post-treatment (Figure
1a, Supplementary Table 4). In patients P01 to P03, baseline clonal architectures
remained relatively stable with proportional changes between parental clones and progeny
subclones on 5-Aza (Figure 1b, Supplementary Figure 2a). In patients P04 to P06, we
saw increasing shifts from parental clones to progeny subclones on therapy (Supplementary
Figures 2b-d). In patients P07 to P10, clonal architecture was characterized by expansion
of maximally mutated progeny subclones on 5-Aza (Figure 1c, Supplementary Figures
2e-f).
We next delineated clonal evolution in patients with CN-LOH. Patient P11 harbored
subclones bearing SRSF2
P95H, RUNX1
L144Q and two CBL variants (CBL
C384Y, CBL
C416Y) at presentation (Figure 2a). While in SD on 5-Aza treatment, the patient acquired
chromosome 11q CN-LOH, with uniparental disomy of CBL
C384Y. These subclones expanded after acquisition of additional RUNX1 mutations and
became dominant at disease progression. A similar pattern of clonal evolution was
observed in patient P12 with CR. At 3 months on 5-Aza, CN-LOH of chromosome 12 led
to elimination of KRASA146V-containing subclones, with reversion to native KRAS; and
focal CN-LOH in chromosome 17 led to reduction of SRSF2
P95H-containing subclones. At 6 months, clonal architecture was largely simplified
to subclones containing TET2
V239fs and SH2B3
V402M (Figure 2b).
Our data illustrate early clonal dominance and clonal heterogeneity with co-existence
of parental and progeny populations at baseline. TET2, SRSF2 and ASXL1 mutations were
detected as co-founding events in individual subclones across different CMML patient
samples, and their VAFs were not altered despite clinical response, as previously
described11. Distinct evolution patterns were observed, ranging from relative preservation
of baseline clonal architecture to expansion and dominance of progeny subclones through
successive acquisition of mutations or via LOH. As an example, in patient P11 with
CN-LOH, disruption of the ring finger domain critical for E3 ligase activity via acquired
biallelic CBL
C384Y mutation correlated with myelomonocytic expansion
12, 13
. Subsequent acquisition of inactivating RUNX1 mutations further enabled dominance
of these CBL
C384Y-bearing subclones. Overall, global suppression of myelomonocytic cells was achieved
after 4 cycles of 5-Aza, with re-expansion of lymphocytes to a median of 24% in patients
with CR or PR (n=6) (Supplementary Figure 3). However, clonal evolution patterns did
not correlate with response to HMAs. Clonal evolution with expansion of maximally
mutated progeny subclones occurred in 5 out of 8 patients with favorable clinical
response, while progeny subclones evolved and expanded with successive acquisition
of secondary mutations or LOH events in 2 out of 3 patients with disease progression.
This suggests that subclones within the CMML compartment continue to evolve irrespective
of clinical response, and that response is governed by complex genetic signals and
epigenetic mechanisms. Consistent with a previous report
11
, our study highlights that current understanding of CMML biology is predominantly
mechanistic, and accurate correlation of specific pathogenic clones with clinical
response has yet to be determined.
Next generation sequencing (NGS) panels are increasingly used for molecular monitoring
in CMML, but VAFs of somatic mutations alone do not adequately reflect clonal heterogeneity.
Single cell sequencing provides the maximum resolution for delineation of clonal architecture,
but is limited by costs and potential allelic bias
14
. Analysis of single cell colonies may be influenced by specific cytokines used in
the cultures. While current clinical practice is to monitor hematologic parameters
and VAFs of mutations in CMML patients, we demonstrate that subclonal hierarchies
and evolution can be delineated at high resolution from standard bulk NGS data using
the SubcloneSeeker computational algorithm. Inclusion of all somatic mutations rather
than only driver mutations is required to solve clonal architecture with high confidence.
This strategy provides deeper insights into the hierarchy of acquisition and distribution
of founding and secondary mutations within individual subclones in CMML, without resorting
to single cell analysis. The predictive accuracy of the computational algorithm has
recently been validated in a similar study for drug-resistant breast cancer subclones,
using single-cell genotyping experiments
15
. Until single-cell sequencing technologies become widely available for routine testing,
computational reconstruction of clonal evolution represents the most dynamic platform
available for delineation of specific mutations and subclones in leukemia, and may
become a useful tool for response monitoring and potentially therapeutic decision
making.
Ongoing clonal evolution despite apparent clinical remission highlights gaps in the
current mechanistic understanding of HMA therapy in CMML. Integration of epigenetic
evolution and the influence of tumor microenvironment into clonality studies may establish
strategies to unravel biological complexity and identify novel therapeutic targets
in CMML.
Supplementary Material
1