Recent Insights Into Chemotherapy-Induced Thrombocytopenia

Introduction

Chemotherapy-induced thrombocytopenia (CIT) is a common hematologic toxicity of myelosuppressive and ablative therapy. Severe or persistent CIT not only has a risk of life-threatening spontaneous hemorrhage, but also may necessitate reduction and/or delay in treatment doses.^[1] Allogeneic platelet transfusions remain the mainstay of treatment for severe or symptomatic CIT, although when to initiate transfusion may be controversial.

Recently, thrombopoietic growth factors have been developed as therapy for thrombocytopenia. Substantial efficacy of the 2 most advanced agents has been demonstrated in immune thrombocytopenic purpura (ITP) and one has also been effective in hepatitis C-related thrombocytopenia. In these trials, the use of thrombopoietic agents has been very tolerable and without significant toxicity to date. Their use in thrombocytopenia following chemotherapy protocols, or in certain marrow failure states, either as prophylaxis or therapy in patients to avoid hemorrhage and reduce use of platelet transfusions, is currently under study. Our current understanding of the physiologic pathways in platelet production and novel strategies under current investigation for management of CIT are described.

Chemotherapy-Induced Disruption of Platelet Production

Platelets are small, anucleate circulating cell particles that are released into the bloodstream by megakaryocytes. As for all hematopoietic progenitor cells, development of megakaryocytes occurs by an intricate cascade of signaling events, organized temporally and spatially by specific cytokines and growth factors.^[2] Thrombopoietin (TPO), the primary regulator of thrombopoiesis, promotes megakaryocyte differentiation from hematopoietic stem cells, acting in conjunction with other cytokines including interleukin (IL)-3, IL-6, and IL-11.^[3] Microenvironmental factors contribute to this process. Interactions between megakaryocytes and bone marrow stromal components are critical for platelet production. Within the bone marrow, stromal-derived factor 1alpha (SDF-1alpha) and fibroblast growth factor 4 (FGF-4) promote migration of megakaryocytes from the endosteal stem cell compartment to the vascular zone, where megakaryocytes adhere to the marrow sinusoids.^[4] Here, interaction with the uniquely specialized bone marrow endothelial cells supports cellular maturation, remodeling, and endomitosis. Once mature polyploid cells, megakaryocytes develop proplatelet formations, with multiple platelet-sized beads along their length, which may then be released into the peripheral circulation. Chemotherapy for malignancy results in depletion of stem and progenitor cells and the bone marrow stroma (myeloablation), with depression of blood formation until the hematopoietic tissue bed repairs and reconstitutes.

Thrombopoietic Growth Factors

Although the presence of a thrombopoietic-specific cytokine was first suggested in 1958, TPO was not cloned until 1994.^[5,6] Given the success of granulocyte colony stimulating factor and erythropoietin therapy in promoting lineage-specific hematopoiesis, TPO agents were anticipated to have great therapeutic potential in CIT and other thrombocytopenic disorders.

Recombinant Human Thrombopoietin Agents

The first agents developed were recombinant forms of human TPO, pegylated recombinant human (rHu) megakaryocyte growth and development factor (PEG-rHuMGDF), and rHu TPO. Trials in healthy volunteers showed increased platelet counts and megakaryocyte mass with minimal toxicity and multiple clinical trials also demonstrated unequivocal effects on the platelet count.^[7] However, further development ceased after intravenous and subcutaneous administration initiated development of neutralizing antibodies that cross-reacted with endogenous TPO and resulted in significant thrombocytopenia in a number of recipients, including previously healthy donors.^[8,9]

Thrombopoietin Receptor Agonists

Focus then shifted to nonimmunogenic, second generation thrombopoietin receptor (c-Mpl) peptide agonists that shared no sequence homology with native TPO.^[10] Two of the c-Mpl agonists, AMG531^[11] and eltrombopag,^[12] are now in phase 3 clinical trials for ITP. AMG531 is also being tested in myelodysplastic syndrome and cytopenias associated with non-Hodgkin's lymphoma and a number of types of cancer. Eltrombopag is also being tested in hepatitis-C-associated thrombocytopenias and other trials in sarcoma, metastatic disease, renal impairment, and hepatic impairment are being initiated.^[11-13]

AMG531 and eltrombopag bind to the human TPO receptor and induce phosphorylation of JAK2 and STAT5 signaling pathways, promoting megakaryocyte differentiation, proliferation, and platelet production. Both have been studied in phase 1-3 trials in adults with chronic ITP. Results of phase 1/2 trials were published recently, demonstrating that AMG531 given as a weekly subcutaneous injection for 1-6 weeks lead to doubling of platelet counts and an increase to more than 50 x10⁹/L in many treated patients with minimal side effects.^[11,14]

Eltrombopag is an orally available small molecule (molecular weight 547 D). In a randomized, double-blind, placebo-controlled trial, platelet counts increased to more than 50 x10⁹/L in 70% and 81% of patients treated with 50 mg and 75 mg daily doses, respectively.^[12] No significant adverse events were seen. A trial of eltrombopag in hepatitis C treatment has demonstrated similar efficacy and lack of toxicity as seen in ITP.^[15]

Both agents appear to be effective at increasing platelet counts and reducing bleeding symptoms. They have both been used for short-term therapy in adults with chronic ITP with apparent minimal toxicity. To date, only AMG531 has been reported for longer term usage. One concern is that a treatment-induced increase in bone marrow reticulin was reported in 2 patients treated with AMG531 that returned to previous levels following cessation of treatment. Bone marrow histology was not regularly examined in the trials with either agent. There are other thrombopoietic agents in development, such as AKR-501, which appear promising in normal volunteers. Further study will reveal the potential of these agents for long-term maintenance therapy, and the utility of these agents in children with ITP.

Utility of Thrombopoietin Therapy in Chemotherapy and Hematopoietic Transplant Recipients

Despite success in nonmalignant thrombocytopenia, the utility of TPO receptor antagonists in attenuating the platelet nadir, avoiding the need for platelet transfusions, and speeding recovery following chemotherapy is less clear-cut.^[16] The initial trials in the second half of the 1990s were performed with PEG-rHuMGDF. In the first study by Basser and colleagues,^[17] PEG-rHuMGDF was administered before the first cycle of chemotherapy and demonstrated dose-dependent increases in platelet count. Results after chemotherapy were more equivocal, with no difference in the depth of the platelet nadir or recovery between placebo and treatment groups, although the nadir occurred earlier in the treated group.^[18] Fannuci and colleagues^[19] reported that PEG-rHuMGDF could mitigate the degree of CIT in nonmyeloablative therapy, although in this study only 1 patient (in the control group) required a platelet transfusion. Therefore, the ability of PEG-rHuMGDF therapy to reduce the requirement for platelet transfusions was not tested.

Following intensive myeloablation in patients undergoing stem cell transplantation, administration of PEG-rHuMGDF or rhTPO showed no effect on platelet count nadir, time to platelet count recovery to above 20x10⁹/L, or number of platelet transfusions.^[20,21] Indeed, the only demonstrable benefit was seen when rhTPO was administered to the stem cell donor, producing an increase in yield of CD34⁺ progenitor cells and a small but statistically significant reduction in time to hematopoietic reconstitution in the recipient.^[22] Similarly, no significant benefit has been shown for therapy with first-generation TPO agents in patients undergoing high-dose induction or consolidation chemotherapy for acute leukemia.^[23,24]

The varying success between higher-dose (myeloablative) and lower-dose (myelosuppressive) treatment protocols suggests either that a threshold number of megakaryocyte progenitor cells is required for response, and/or the intensive myeloablative regimens also disrupt bone marrow microenvironmental elements, such as the endothelium, that are important for megakaryopoiesis and thrombopoiesis. In addition, levels of endogenous TPO are very high following complete myeloablation, which may limit additional benefit from exogenous growth factor therapy. This may possibly be circumvented with second-generation agents, some of which interact differently than TPO with the TPO receptor.

Implications of Thrombopoietic Growth Factor Therapy for Platelet Harvesting

The pharmacologic interest in thrombopoietic growth factors and cytokines also has implications for transfusion medicine.^[25] TPO receptor stimulating agents may be used to increase the yield of platelets from either healthy donors or patients for autologous harvesting.^[7,26] Thrombopoietic therapy, together with improved storage and preservation of platelet products from autologous donors, may improve platelet transfusion therapy by preventing platelet refractoriness.^[27]

Immature Platelet Fraction: An Indicator of Need for Platelet Transfusion

Reliable prediction of platelet recovery would enable a more judicious use of prophylactic platelet transfusions and possibly of thrombopoietic agents. This requires insight into thrombopoietic state. The proportion of newly produced, 'reticulated platelets' to total platelets reflects the rate of thrombopoiesis and can be quantified with flow cytometry using RNA-binding fluorochromes, such as thiazole orange. The Sysmex-XE2100 (Diamond Diagnostics, Holliston, Massachusetts) now enables automated assay of the immature platelet fraction (equivalent to the reticulated platelet fraction) as part of the routine blood cell count analysis, using a polymethine RNA fluorescent dye.^[28] An increase in the immature platelet fraction (IPF) percentage appears to herald platelet recovery in patients undergoing cytotoxic chemotherapy and following autologous stem cell transplantation,^[29] suggesting that prophylactic platelet transfusions for patients without bleeding are probably unnecessary if an increase in IPF is detected, because the platelet count will recover in 1-2 days.

Platelet Growth Factor and Cytokine Therapy in Chemotherapy-Induced Thrombocytopenia: Future Directions and Unanswered Questions

Preclinical studies demonstrated that following 5-fluoruracil treatment of transgenic mice deficient in the TPO/c-Mpl axis, administration of cytokines SDF-1alpha and FGF-4 reconstituted thrombopoiesis, even in the absence of TPO signaling.^[4] This effect was mediated by these cytokines promoting megakaryocyte migration to and adherence at the vascular niche.^[2] Additional findings in murine models that underlie the importance of the endothelial compartment for hematopoietic reconstitution indicate that infusion of ex-vivo expanded endothelial progenitor cells accelerates hematopoietic recovery following sublethal irradiation.^[30] Remarkably, the endothelial progenitors administered were not detectable in either bone marrow or blood; therefore, these effects must occur by indirect mechanisms, the details of which remain unclear. Further elucidation of the molecular and cellular interactions between the megakaryocyte and the endothelial cells that are necessary for thrombopoiesis may lead to therapeutic advances in growth factor therapy for CIT. In the arena of transfusion and transplantation medicine, growth factor therapy may be used to augment stem cell/progenitor harvesting, and to expand megakaryocyte progenitors ex vivo to supplement stem cell therapy.

Summary

Recent insights into megakaryocyte physiology and the biogenesis of platelets have led to significant therapeutic advances in the treatment of benign thrombocytopenia. Clinical trials have shown that use of thrombopoietic growth factors is practical and efficacious for nonmalignant thrombocytopenia and following nonmyeloablative chemotherapy regimens. Further clarification of the precise roles for growth factors, cytokines, and bone marrow microenvironmental components is required to understand how to improve the limited success of these agents following myeloablation. Preclinical data suggest that the bone marrow vasculature plays an integral role in thrombopoiesis, and strategies to expedite recovery of these bone marrow cells following chemotherapy and the appropriate use of chemokines in conjunction with thrombopoietic agents may prove beneficial.

Acknowledgments

Bethan Psaila is a Fulbright Scholar in Cancer Research and a recipient of a Kay Kendall Leukemia Fund Traveling Fellowship, and this work was partly supported by NIH grant U01 HL072196 (Dr. Bussel), Dana Hammond Stubgen, and the Children's Cancer and Blood Foundation.

Supported by an educational donation provided by Amgen.

References