Sunday, May 4, 2014

Pre-leukemic HSCs bearing mutated DNMT3A undergo multilineage differentiation and evade chemotherapy

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 CD33cell 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.

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 (Shlush, Liran, Sasan Zandi, et al. 2014)

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.

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.


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.