10 subjects provided paired pre-treatment and day 4 samples for flow cytometric analysis. The baseline characteristics of subjects who provided paired flow data is shown in . The median day 4 trough rapamycin concentration for these subjects was 8.6 ng/mL (range 3.5–12.8). Day 7 trough rapamycin concentrations were slightly higher (mean 12.6 ng/mL, range 4.8–22.1).
Baseline characteristics of the ten subjects who provided paired samples for flow cytometric pharmacodynamic analysis.
Previous work (reviewed in (19
)) has demonstrated that the CD45 by side scatter plot allows cells to be divided into four cell populations: lymphocytes, monocytes, progenitor cells/granulocytes, and blasts. AML cells typically cluster within the blast gate (CD45 dim, side scatter low), although acute myelomoncytic/monoblastic leukemic blast poupulations may extend into or exclusively occupy a CD45 bright region otherwise enriched for monocytes.
Although our cell processing reduced the brightness of cell surface antigen staining to some degree, it nonetheless provided excellent discrimination of light scatter and CD45 expression. This was sufficient to define discrete cell population gates in all subjects (, top left). Of note, 4/10 subjects on our study had a total white blood cell (WBC) count below 5 × 103/μL and an absolute blast counts (ABC, defined as blast percentage × total white blood cell count) below 1 × 103/μL on at least one pharmacodynamic peripheral blood draw. An additional subject had no visible circulating blasts by peripheral smear and underwent paired marrow aspiration at baseline and day 4 for pharmacodynamic sampling. Morphologic hematopathology review of the marrow aspirate in this subject showed only 15% blasts. Depending upon the subjects’ clinical immunophenotypes, antibodies directed against myeloid and/or immaturity surface antigens (e.g. CD33 and CD34) were used to confirm the blast gate and were informative in all cases. These findings demonstrate the feasibility of obtaining consistent, tumor-specific data on peripheral blood samples even on patients with a low WBC.
Identification of blast gate and establishment of positive gates for phospho-S6
S6 phosphorylation was evaluable in all subjects samples, even in cases where the WBC was as low as 1.1 × 103 with 10% blasts (i.e. ABC= 110/μL). In several subjects with acute monocytic or myelomonocytic leukemias, expansion of both a CD45 dim, SSC low blast gate as well as a CD45, SSC-intermediate monocytic gate was noted. In these cases, each population was evaluated for S6 phosphorylation. Discrimination between monocyte and lymphocyte populations was made based upon CD33 staining and both blast and monocyte population analysis was performed in these cases (, top right). Thus, we were able to analyze S6 phosphorylation in separate populations of normal and malignant cells in all samples.
Comparing samples drawn at baseline to samples treated ex vivo with PMA or rapamycin, we saw dynamic changes in S6 phosphorylation (, bottom). In all subjects’ samples, PMA led to marked upregulation of S6 phosphorylation in all CD45+ cell populations, regardless of staining pattern in the unmanipulated sample. This serves as an internal control for cell viability and served as a positive control for the purpose of estimating the percentage of blasts with mTOR activation at baseline. It further shows that the phospho-S6 epitope is fully unmasked by our cell processing method and capable of resolution if phosphorylated. Ex vivo treatment with ≥500 nM rapamycin markedly reduced or abrogated S6 phosphorylation in all samples that showed baseline constitutive S6 phosphorylation. Comparing PMA as a positive control and rapamycin and/or FMO treatment as positive and negative controls, respectively, we created discrimination lines for positive and negative phospho-S6 populations and scored percentage of positive cells for each group of cells.
For baseline samples, the most consistent finding was marked heterogeneity of S6 phosphorylation amongst leukemic blasts. The majority of samples (8/10) showed both a dominant, discrete population that lacked S6 phosphorylation (similar phospho-S6 brightness to either the FMO control or lymphocytes) and a smaller subset of cells with heterogeneous, but constitutive S6 phosphorylation, comparable in brightness to the range of PMA stimulated blasts. In two subjects’ baseline samples, phospho-S6 staining fell uniformly in the negative gate and no obvious positive phospho-S6 events were seen. From these gates, we defined baseline percentages of positive cell events. In cases where there was marked separation between narrowly clustered positive and FMO populations, a midpoint between the two populations was chosen for discrimination, as previously described.(20
) These data demonstrate that, consistent with previous results from our lab and others, mTOR is constitutively activated in AML blasts in the majority of primary samples. However, this activation is actually only present in a subset of blasts at any one time.
The described intracellular phospho-specific flow cytometry of fixed whole blood allowed for several applications. For example, we used the methodology to determine the sensitivity of AML cells to rapamycin. To do this, we evaluated the effect of a 30 minute ex vivo incubation of the baseline sample in increasing concentrations of rapamycin to estimate a dose-response curve for mTOR inhibition in leukemic blasts. A representative sample showing low nanomolar sensitivity to rapamycin is shown in (top). Note that concentrations below 10 nM (corresponding to clinically measured concentrations of 9.1 ng/mL) fail to produce substantial reduction in S6 phosphorylation, but concentrations at or above 20 nM show similar reduction to that of 1000 nM rapamycin. This suggests that near-maximal inhibition occurs between 10–20 nM in this sample, which is similar to clinically achieved concentrations. These studies demonstrate that we can assay in vitro drug sensitivity on AML cells and compare the effects of in vitro inhibition of target with in vivo results. This allows us to discriminate between patients who received inadequate concentrations of drug in vivo compared to samples with endogeneous rapamycin insensitivity.
Ex vivo inhibition of mTOR predicts in vivo effects of oral sirolimus therapy
Flow cytometry also allowed for analysis of signaling in different subpopulations of blast cells. In one subject with AML arising from underlying chronic myelomonocytic leukemia, the baseline sample demonstrated abnormal expansion of both an immature myeloblast population as well as a more differentiatied monoblast population by CD45 and side scatter (Supplementary figure 1
). These two populations were confirmed as discrete populations by CD34, CD33, and CD14 staining (CD34+, CD33 dim+, CD14-myeloblasts vs. CD34-, CD33 bright+, CD14+ monoblasts). Interestingly, the myeloblast population showed no baseline phosphorylation of S6, though constitutive phosphorylation was readily seen in monoblasts. 30 minute ex vivo
treatment with rapamycin failed to potently inhibit S6 phosphorylation in monocytes until concentrations above 50 nM were evaluated. Notably, 50 nM exceeds measured blood rapamycin concentrations from the trial. Thus, we are methodologically able to demonstrate variations of signaling within sub-populations of leukemic cells, which may be important for understanding the diverse response of leukemia to signal transduction inhibitors.
Finally, we focused on clinical evaluation of the effect of oral sirolimus therapy upon S6 phosphorylation (, bottom). Because the majority of the blast events showed phospho-S6 staining in a range comparable to FMO, shifts in the mean fluorescence intensity were less dramatic than changes in the percentage of cells in the positive phospho-S6 gate. To quantify the degree of mTOR inhibition, we calculated an inhibition percentage, defined by 1 minus the fraction of positive phospho-S6 blasts on day 4 divided by the percentage at baseline. Thus, if a subject had 20% phospho-S6 positive blasts at baseline and 5% on day 4, the inhibition percentage was 1- (5/20)]= 75% inhibition. We therefore could use changes in S6 phosphorylation during therapy to group subjects based upon their blasts’ biochemical sensitivity to sirolimus.
Three patterns of baseline mTOR activation and response to sirolimus emerged from our anaysis. Of the 8 subjects that showed baseline constitutive phosphorylation of S6, 6 showed marked reduction in the percentage of phospho-S6 positive events () Such subjects blasts were considered biochemically “sirolimus-sensitive” and showed a mean inhibition percentage of 75% reduction in the percentage of cells in the positive gate for S6 phosphorylation (range 53–98%) The two subjects without constitutive phosphorylation of S6 showed no appreciable change in their blasts’ S6 phosphorylation on day 4 and were considered uninformative for sirolimus sensitivity. Two subjects showed persistent S6 phosphorylation in their blasts on day 4 and were considered sirolimus-resistant. In one subject, baseline and day 4 percentage positive phospho-S6 events were similar and in the other a marked increase in phospho-S6 events was seen. Of note, this subject’s baseline sample was noted to require relatively higher concentrations of rapamycin in ex vivo testing to demonstrate inhibition of S6 phosphorylation. Interestingly, measured sirolimus troughs were similar in biochemically sirolimus sensitive and resistant leukemia. Overall, 8/10 (80%) of patients demonstrated activation of S6 at baseline. Of these 8 subjects 6/8 (75%) were inhibited in vivo. One of these two patients demonstrated sirolimus resistance both in vivo and in vitro. The other subject’s samples were not evaluated for in vitro sensitivity. 4 subjects had ex vivo testing performed on their baseline samples. 3/3 subjects whose ex vivo testing of their leukemia sample for rapamycin sensitivity demonstrated sensitivity at ≤20 nM had biochemical sensitivity to sirolimus during therapy. One subject baseline showed no inhibition of S6 phosphorylation ≤20 nM, modest reduction at 50 nM, and marked inhibition at 1000 nM. This subject’s day 4 sample showed biochemical sirolimus resistance.
Figure 4 Patterns of AML blasts’ pharmacodynamic responses to oral sirolimus therapy Subjects’ samples showed either constitutive S6 phosphorylation at baseline (n=8) or no obvious phosphorylation at baseline (n=2). The effects of 72 hours of oral (more ...)
Although the intent of this pilot study was not to evaluate clinical efficacy, clinical responses to sirolimus and MEC were evaluable in 9 of 10 subjects who provided paired samples for flow analysis. One subject died of bacterial infection prior to hematopoietic recovery from sirolimus MEC and was not evaluable for treatment response. Among the 5 evaluable sirolimus-sensitive subjects, 1 CR and 2 PRs were seen. Neither of the 2 evaluable sirolimus-resistant patients showed a clinical response. One of the two evaluable subjects without baseline S6 phosphorylation had a PR.