It is still unclear how to effectively assess whether leukemic stem cells are actually being targeted. As new targeted agents enter clinical trials, the solution to this problem becomes more pressing. In addition, candidate agents may be more or less effective at targeting leukemia stem cells based on the patient population: whether patients have achieved remission and the targeted agent is used as maintenance therapy, or whether patients are relapsed or refractory ().
Potential use of leukemia stem cell (LSC)-targeted agents
A targeted agent could be used as part of a maintenance regimen to destroy the residual leukemic stem cells in patients in remission. It is an attractive concept to think that a small residual amount of disease contains the stem cell compartment. The concept is particularly appealing if a targeted agent can be used in a maintenance regimen to eliminate the last bit of disease, especially if the targeted agent has a relatively low toxicity and can be used for a long period of time. However, there is no evidence that any of the agents currently available can target leukemia stem cells in a remission patient. The biology of cells in minimal residual disease conditions appears to be very different from that of cells found in a de novo and heavy tumor-burden context. This difference can impact whether the drugs work or not. As clinical trials progress, the targeted agents may fail because of the physiology of the tumor cells, not because the drug is ineffective with all leukemic stem cells.
Cancers are heterogeneous, and this is exceptionally true of leukemic stem cells. From patient to patient, molecular markers for stem cells differ greatly. In some patients, the leukemic stem cell may have a particular pattern of phenotypic markers such as CD38 and CD34, but other patients can have very different expression levels and frequencies of the same antigens. This heterogeneity makes determining the frequency of the leukemic stem cell in an individual patient extremely difficult. Similarly, during treatment, the phenotype and the genetics of the leukemic stem cell are also likely to be highly unstable. During chemotherapeutic challenge, patients can experience a dramatic change in the phenotype of their leukemic stem cell. The candidate leukemic stem cell population in a de novo patient at presentation may or may not be present after treatment. This phenotypic change suggests that the genetics of these stem cells are also probably changing. What we define as a leukemic stem cell is a highly dynamic and highly unstable entity in the context of any patient population and probably varies as a consequence of therapeutic regimen. This makes the overall monitoring and analysis of these populations quite challenging.
Parameters for analysis of leukemic stem cell in clinical trials
Current standard clinical endpoints may not be informative to evaluate the tumor in patients with relapsed or refractory disease. A reduction in total tumor burden may or may not be indicative of what is happening at the stem-cell level. Consequently, investigators must be able to identify and quantitate leukemic stem cells in any kind of clinical context.
To do so, a number of steps can be taken. Clinical trials must be temporal and patient-specific. Specimens must be gathered before, during, and after treatment. The phenotype of each patient’s leukemic stem cell population must be defined up front and verified by a functional assay to help quantitate it. In the course of treatment, the population must be continuously monitored, as has been done successfully by Gerrit Jan Schuurhuis and colleagues.14
The panel of antibodies must be customized for each patient because each patient may have a different leukemic stem cell phenotype. Some antibodies that appear to be useful in terms of defining leukemia stem cells are CD34; CD38; and CD123, the IL-3-receptor alpha chain.15
and CD47 (I. Weissman, unpublished) have also been reported to be upregulated on leukemia stem cells.
Ideally, a molecular marker will be present. Molecular markers, like NPM and FLT3, are present in a large percentage of AML patients. For many patients, these molecular markers can be used in residual disease to determine overall tumor burden.
Though a very well-refined phenotypic panel that uses multiple markers is a good surrogate, a functional assay is the only rigorous way to define the stem cell population. Therefore, employing a quantitative functional analysis will be a very important and very difficult practical aspect of determining whether the leukemic stem cells are being targeted. During treatment, the ability for progenitors to form colonies in vitro or for leukemic stem cells to engraft NOD/SCID mice in vivo will be a critical element of determining whether or not the markers are correctly identifying stem cells. This will certainly be challenging, especially in the context of residual disease patients in remission, but it must be attempted.