In my previous post, I introduced Hematopoietic Stem
Cells (HSCs) and their involvement in acute myeloid leukemia (AML). I mentioned
a very important finding from Shlush’s paper in Nature that mutated DNMT3A is present in the
pre-leukemic ancestral cells which give rise to the T cells and AML cells found
at the time of diagnosis of the disease.
I
now go further to discuss the types of non-leukemic HSC populations in which the
mutated DNMT3A first arises.
Researchers isolated non-leukemic hematopoietic stem and progenitor cell
populations from 11 mutated DNMT3A/NPM1c AML
patients. Note that progenitor cells are relatively immature cells that are
precursors to a fully differentiated cell of the same tissue type. The cells
isolated from these patients were the hemotopoietic stem cells/multipotent
progenitors (HSCs/MPPs), multilymphoid progenitors (MLPs), common myeloid
progenitors (CMPs), granulocyte monocyte progenitors (GMPs), megakaryocyte
erythroid progenitors (MEPs), as well as mature B, T and natural killer (NK)
cells within a CD33- cell fraction and CD33+ AML blasts. These were highly
purified, phenotypically normal cell populations that were assessed by ddPCR
for allele frequencies of DNMT3A and NPM1c,
both of which were found together in CD33+ AML blasts but DNMT3A was also found alone at variable allele frequency (mean is
24.6%) across the spectrum of mature and progenitor cells.
The
data below is for a single, representative patient no. 11 whose cells were examined
for DNMT3A and NPM1c at three particular times: diagnosis, remission (3 months)
and relapse to evaluate the role that mutated DNMT3A plays in the progression of the disease.
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Figure 2b. Allele frequency of DNMT3A and NPM1c mutations in stem/progenitor, mature lymphoid and blast cell populations, as indicated, isolated from diagnosis (blue), remission (white) and relapse (red) samples of patient no. 11 as determined by droplet digital PCR (
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In this sample, the mutated DNMT3A in HSCs/MPPs was found at an allele frequency of 12-30%
without mutated NPM1c, which
according to the authors, is a high enough mutated DNMT3A frequency to have a significant clonal contribution as
compared to non-mutated HSCs. It was
also found that compared to diagnosis, the allele frequency of mutated DNMT3A alone was similar to or higher at
remission and relapse. What’s interesting is that CD33+ leukemic blasts always
contained both mutated DNMT3A and NPM1c at diagnosis, but CD33+ myeloid
cells at remission contained only DNMT3A ,
indicating that they were not AML blasts but progeny of the mutated DNMT3A bearing progenitor cells with
preserved ability to differentiate into myeloid lineage. At relapse, both
mutations are again present in majority of the cells of patient no. 11 with the
exception of HSCs/MPPs, in which a proportion contained only mutated DNMT3A alone.
The figure above is cell sample data from a single
patient who the researchers believe accurately represents the data from five of
the original eleven mutated DNMT3A/NPM1c AML
patients examined in this study. Although the patient’s allele frequencies are
representative of that of the other individuals, I believe that the researchers
should have collected data from a larger sample of AML patients and reported
the allele frequencies in the mutated DNMT3A/NPM1c
as an average and standard error of the mean (SEM) to describe the
variability within the sample as it is reported this way in many medical
journals.
Another patient no. 57 was a long-surviving patient
so cells from both early and late (36 months) remission could also be examined.
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| Figure 2d. DNMT3A mutant allele frequency in sorted cell populations isolated from diagnosis (0 months), early (3) and late (36) remission samples of patient no. 57 (Shlush, Liran, Sasan Zandi, et al. 2014) |
There is a significant increase in mutated DNMT3A allele frequency in most cell
populations over time (see Figure 2d) . In addition, though not evident in the figure, a small
proportion of CD33+ myeloid cells at late remission also contained both mutated
DNMT3A and NPM1c found in diagnosis. This meant that somehow there had been a
regrowth in the diagnostic leukemic clone or emergence of a new clone following
an independent NPM1c mutation within
the pool of pre-leukemic ancestral cells. This suggests that the mutated DNMT3A found in HSCs/MPPs at diagnosis
was capable of multilineage differentiation that somehow evaded chemotherapy
and gave rise to clones that presented themselves at remission, and could potentially
serve as a “reservoir” for further evolution of these clonal cells that leads
to relapse of the disease.
With this figure, the researchers clearly meant to
emphasize the general trend that shows an increase in the mutated DNMT3A allele frequency across most cell
populations. However, the researchers fail to mention why in particular cell
populations, the allele frequency in the gene increases, decreases or remains
the same over the time period. For example, there is no explanation as to why
there is no noticeable change in the frequency from 0 to 3 months in CMPs and
NKs or why in B cells, the frequency first decreases and then increases in late
remission, whereas in T cells, it only increases. Perhaps the individual
effects of mutated DNMT3A in
individual cell populations are not greatly relevant to the author’s main
argument, which is that the overall frequency increase in this gene contributes
to the relapse of AML. Nonetheless, as with the previous figure, the
researchers could have improved upon this graphic representation by using a
larger sample size, although there could still be the chance that only one
patient survived long enough out of the 50 or so individuals studied to be able
to contribute to the data in this figure.
In terms of treatment, I believe the next logical
step for oncologists should be to continue to monitor the remission state and
to initiate more aggressive therapy as early as possible to target the
population of pre-leukemic HSCs/MPPs before these cells get a chance to acquire
any more mutations or differentiate into leukemic blast cells. I will talk more
about treatment and clinical trials in my next post. Stay tuned.
References:
Shlush, Liran, Sasan Zandi, et al.
"Identification of pre-leukaemic haematopoietic stem cells in acute
leukaemia." Nature. 506.February (2014): 328-33. Web. 13 Apr.
2014.
