A major challenge in the development of anti-cancer strategies that induce differentiation and potentially target cancer stem cells is accurately evaluating and classifying the clinical activity. Using standard AML or MDS response criteria is problematic as they largely reflect changes in total tumor bulk following cytoreductive therapy. Paradoxically, increased tumor burden due to an expansion of mature tumor cell compartments, such as ATRA syndrome in APL, may be an early indicator of successful differentiation activity. In our current study, the mean weighted WBC and ANC, but not the peripheral blasts, significantly increased during the treatment period. It is unclear whether this increase was due to differentiation of malignant progenitors or improvement of normal hematopoiesis. Nonetheless, this quantitative increase was also functional as it appeared to limit the infectious complications of neutropenia since none of the study participants treated in the highest 3 dosing cohorts (18 µg/m2
× 14 days, 20 µg/m2
× 14 days, 16 µg/m2
× 21 days) experienced febrile episodes over 18 cumulative cycles. Importantly, improvements in ANC and febrile episodes were not limited to patients with objective responses in bone marrow blasts or changes in transfusion needs suggesting that bryostatin-1mayhave clinical activity at doses that do not suppress normal hematopoiesis [15
It is also possible that the improvement in blood counts, namely neutrophils, was due to activity of GM-CSF alone rather than the combination. However, based on known similar effects on both normal and malignant cells, one might expect to see a concurrent increase in peripheral blasts along with the higher total white cell count. We did not observe these effects even in patients with active AML at the time of treatment suggesting that the combination primarily impacts normal myeloid growth and maturation either through the inhibition of the malignant clone or direct enhancement of normal hematopoiesis.
The kinetics of clinical responses may also differ between traditional cytotoxic agents and those targeting differentiation pathways. Limiting the self-renewal capacity of leukemic stem cells would likely not produce an immediate or dramatic response, but rather a more delayed and gradual one as the production of new tumor cells was inhibited. Although the anti-leukemic effects of bryostatin-1 and GM-CSF were modest during treatment, two of the patients treated at the highest doses of bryostatin-1 experienced durable improvements in both leukemic blast counts and normal peripheral blood counts. Interestingly, these improvements were not immediate but gradual in nature following the discontinuation of the treatment due to toxicity. Importantly, these responses were prolonged with the one CR lasting for approximately one year and the second patient free of transfusions for 18 months. Although far from definitive, these results suggest that differentiation targeted therapies may target leukemia stem cells in a clinically meaningful fashion.
The pharmacology of bryostatin-1 administered by continuous infusion is complex and limited by availability of sensitive analytical techniques [23
]. While our analytical method could measure bryostatin-1 levels as low as 50 pg/mL [26
], a more sensitive analytical method should be developed in order to understand this agent’s clinical pharmacology. We were able to assess whether biologically relevant levels of drug were reached in patients through an in vitro
correlative study using patient plasma pre- and during treatment on a cell line known to be sensitive to bryostatin-1. Even though doses of bryostatin-1 under 12 µg/m2
were not measurable in patient plasma samples with LC/MS/MS, the correlative assays demonstrated that drug levels in the patient plasma samples could suppress cell growth. This clonogenic growth suppression appeared to occur in a dose–response fashion in samples studied. Using a control dose–response curve, drug levels were extrapolated by quantifying clonogenic recovery and showed that continuous infusion of bryostatin-1 was effective at suppressing 40% of colony growth at doses equal to or greater than 16 µg/m2
. Such studies are helpful in providing secondary evidence of drug activity and may also reflect the impact of biologic agents on residual and small populations of disease. In addition to these studies, efforts to quantify leukemia stem cells serially should be undertaken and further developed to be incorporated more broadly into differentiation based clinical trials.
Unfortunately, there were significant toxicities seen with this combination. Consistent with previous clinical trials involving bryostatin-1, myalgias and arthralgias were the predominant dose-dependent adverse events that precluded participants from tolerating maximal dose escalation [29
]. However, the addition of growth factor did not appear to worsen or lessen these expected bryostatin-1 toxicities. Though the toxicities of bryostatin-1 limit its further development, this combination serves as a template for new studies combining growth factors with pharmacologic differentiation agents that offer better side effect profiles. Future studies will benefit from efforts to integrate correlative studies, such as the serial measurements of leukemic stem cells, and together may determine whether approaches targeting differentiation ultimately inhibit these clonogenic cells.