- Email: [email protected]
Autologous bone marrow mononuclear cell therapy for critical limb ischemia is effective and durable
From the Midwestern Vascular Surgical Society
Autologous bone marrow mononuclear cell therapy for critical limb ischemia is effective and durable Tiffany W. Liang, MD,a Andrea Jester, MD,a Raghu L. Motaganahalli, MD,b Michael G. Wilson, PhD,c Patricia G’Sell, RN,d George A. Akingba, MD, PhD,b Andres Fajardo, MD,b and Michael P. Murphy, MD,b Indianapolis, Ind Objective: We have previously shown that autologous bone marrow mononuclear cell (ABMNC) therapy improves measures of limb perfusion, rest pain, wound healing, and amputation-free survival (AFS) at 1 year in patients with critical limb ischemia (CLI). Long-term durability of ABMNC therapy for CLI remains unknown. The objective of the current study was to evaluate long-term clinical outcomes 5 years after treatment. Methods: Data were retrospectively gathered from a database and via a patient survey and review of medical records of patients previously enrolled in this phase I/II trial. AFS, freedom from major amputation, and freedom from major adverse limb events (MALE) were calculated using the product-limit estimate. The incidence of cardiac, malignant, and other medical events relevant to the safety of cell therapy were tabulated during the time from treatment to follow-up. Results: Twenty-one of the 24 patients (88%) who completed the initial 1-year phase I/II trial were available for the 5year analysis; AFS was 74% (95% confidence interval [CI], 0.53-0.87), freedom from major amputation was 78% (95% CI, 0.58-0.90), and freedom from MALE was 65% (95% CI, 0.45-0.80). Three patients (14%) had major cardiac events. There were no incidences of malignancies or diagnoses of clinically significant proliferative retinopathy. Fifteen patients (71%) report continued improvement in pain-free walking. Nineteen (90%) patients believed that the study was of significant medical value and would participate again. Conclusions: ABMNC therapy provides long-term freedom from AFS, major amputation, and MALE that are comparable with other reports of patients who underwent surgical and endovascular interventions for CLI. Furthermore, no patients developed tumorigenesis or clinically significant retinopathy. Because of the limited number of patients studied, our findings will need to be followed up in a larger phase III trial. (J Vasc Surg 2016;63:1541-5.)
Critical limb ischemia (CLI) is a debilitating condition characterized by rest pain, ulcers, or gangrene due to chronic ischemia.1 There is an estimated 500 to 1000 newly diagnosed cases of CLI per every 1 million people in Europe or North America.1 Current treatment includes
From the Department of Surgery,a Division of Vascular Surgeryb Division of Clinical Pharmacology,c and Indiana Center for Vascular Biology and Medicine,d Indiana University School of Medicine. Biomet Biologics, LLC (Warsaw, Ind) provided the MarrowStim cell isolation system for use in the initial study. They also provided the data for Fig 1. Otherwise, Biomet Biologics, LLC had no involvement in the study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. Clinical trial registration number NCT00113243. Author conflict of interest: none. Presented at the Thirty-fifth Annual Meeting of the Midwestern Vascular Surgical Society, Chicago, Ill, September 15-17, 2011. Correspondence: Michael P. Murphy, MD, Division of Vascular Surgery, Department of Surgery, Indiana University School of Medicine, 1801 Senate Blvd, MPC# 2-3500, Indianapolis, IN 46202 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2016 by the Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvs.2016.01.022
surgical or catheter-based revascularization procedures; medical and pharmacologic therapies can be used in “no option” patients (ie, if revascularization is not possible).1,2 Among these “no option” patients, approximately 40% will require a major amputation and up to 20% will have died within 6 months.1 Because of the high rate of morbidity and mortality for CLI patients who are unable to undergo successful reperfusion, there has been extensive research into alternative treatment strategies. These include gene therapy with vascular endothelial growth factor3,4 and hepatocyte growth factor5,6 as well as cell therapy with bone marrow-derived cells.7-11 The rationale behind these therapies is facilitation of angiogenesis in the ischemic limb. We have previously published 12-month results from an open-label, phase I/II clinical trial involving intramuscular injection of autologous bone marrow mononuclear cells (ABMNCs) into the affected limb, where we reported that this mode of therapy is safe and effective for increasing amputation-free survival (AFS),12 and on the basis of this information we have proceeded with a pivotal multicenter, randomized, double blinded phase III study, the MarrOwStim Peripheral Arterial Disease Kit for the Treatment of Critical LimB IschemIa in Subjects with Severe Peripheral ArteriaL DiseasE (MOBILE) trial. However, for cell therapy to be considered a viable option for longterm management, any beneficial effects should withstand 1541
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the test of time. Because there are few studies in the literature that show outcomes beyond 3 years after therapy, the objective of this report was to follow-up our previous study and determine if the initial promising results held up 5 years later. METHODS Patient selection. A complete description of initial patient selection and characteristics has been published previously.12 In brief, 29 patients (30 limbs) between the ages of 21 and 85 years with “no option” CLI from different parts of the United States were enrolled between September 2005 and March 2009. These patients had symptoms of rest pain and/or ulceration (21 limbs had rest pain only, six had rest pain and ulceration, and three had ulcers only) with an ankle brachial index #0.55 and/or toe brachial index #0.40, as well as an absence of a patent artery below the knee that was continuous to the foot, confirmed using contrast arteriography. An independent vascular surgeon had determined that bypass and/or endovascular approaches to improving perfusion were not possible options for management. As indicated previously, the institutional review board approved the trial protocol, and all patients provided written informed consent to participate in the study.12 Of the initially enrolled 29 patients, two patients died and three limbs were either lost to follow-up or had reached an end point by the 1-year time point. The remaining 24 patients (24 limbs) continued to be included in the study for long-term follow up. Study design. A detailed study design has been presented previously.12 In brief, this was a phase I/II openlabel, prospective study that consisted of a single-dose injection of ABMNCs isolated by using either conventional Ficoll density centrifugation (first 14 patients enrolled) or the MarrowStim (Biomet Biologics, Warsaw, Ind) pointof-care centrifugation system (15 patients, 16 limbs). The ABMNCs were injected intramuscularly to the affected limb distal to the anterior tibial tuberosity. The MarrowStim (Biomet Biologics) system is an alternative cell isolation method that maintains sterility and takes <20 minutes to perform, tested in this study to replace the traditional Ficoll system. These two isolation systems were found to yield comparable ABMNC recovery cell counts; as published previously, the average numbers of ABMNCs obtained after Ficoll and MarrowStim (Biomet Biologics) isolation were 1.3 6 0.7 109 and 2.0 6 1.6 109 cells, respectively (Fig 1).12 The initial study subjects that remained continued to be followed to examine long-term outcomes at 5 years. To this end, we sent out questionnaires, conducted telephone interviews, and retrospectively reviewed their medical records. Outcome measures. The primary outcome was AFS, defined as no major amputation and no death. Secondary outcomes were freedom from major amputation and
Fig 1. Comparison of autologous bone marrow mononuclear cell (ABMNC) recovery from Ficoll and MarrowStim (Biomet Biologics, Warsaw, Ind) isolation systems. Data from Biomet Biologics, LLC.
Table. Censoring mechanisms for three time-to-event outcomes Outcomes AFS Freedom from major amputation Freedom from MALE
Events Death Amputation Amputation Amputation MALE
Censoring mechanisms Lost to follow-up Study termination Death Lost to follow-up Study termination Death Lost to follow-up Study termination
AFS, Amputation-free survival; MALE, major adverse limb events.
freedom from major adverse limb events (MALE). Major amputation was defined as any amputation proximal to the malleolar level, such as a below-knee or above-knee amputation. MALE was defined as any major amputation, minor amputation including transmetatarsal amputation, or surgical reintervention (eg, angioplasty, stenting, or bypass). However, because all subjects were classified in the “no option” designation, none received surgical intervention after study initiation. Therefore, MALE was effectively any major or minor amputation. Additionally, to examine long-term safety, we tabulated the incidence of major adverse cardiac events, malignancies, and clinically significant proliferative retinopathy that might have occurred from the time of ABMNC injection to follow-up. Major adverse cardiac events were defined as Q-wave myocardial infarction, stroke, or death. Malignancy was defined as biopsy-proven malignant transformation of a lesion. Statistical analysis. A summary of events and censoring mechanisms for each outcome is provided in the Table. The product-limit estimates13 for each outcome and the associated 95% confidence intervals (CIs) were calculated. The upper and lower bounds of the 95% pointwise CI only exist at clinical failures and are undefined for censored observations. Using these product-limit estimates, the survival curves for each of the three outcomes were plotted. Under each plot,
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Fig 2. Kaplan-Meier curve of 5-year amputation-free survival (AFS).
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Fig 4. Kaplan-Meier curve of 5-year freedom from major adverse limb events (MALE).
experienced major cardiac events. None reported malignancies or diagnoses of clinically significant proliferative retinopathy. Other than adverse events, 15 patients (71%) reported continued improvement in pain-free walking on the basis of a walking impairment questionnaire. None reported deterioration of walking ability. When asked the question, 19 patients (91%) believed that the study was of significant medical value and would participate again. DISCUSSION
Fig 3. Kaplan-Meier curve of 5-year freedom from major amputation.
the number of limbs entering the interval at risk is annotated under the label of the major tick mark. RESULTS Consolidating previous knowledge of subjects from the initial 1-year study and new information gathered from the questionnaires and telephone interviews at 5 years, we compiled the results. Twenty-one of the 24 patients (88%) who did not die or drop out of the 1-year phase I/II trial responded to follow-up. Of the original 29 subjects (30 limbs), two patients had died (one from suicide), four limbs had minor amputations, two limbs had major amputations, and three limbs were either lost to followup or had reached an end point by the end of the 1-year study. By the 5-year time point, three additional limbs underwent major amputations. At 5 years, AFS was 74% (95% CI, 0.53-0.87; Fig 2); freedom from major amputation was 78% (95% CI, 0.58-0.90; Fig 3); and freedom from MALE was 65% (95% CI, 0.45-0.80; Fig 4). Regarding the 5-year incidence of adverse events, three of the 21 subjects who had follow-up at 5 years (14%)
The natural history of CLI frequently involves need for amputation, major cardiovascular events (eg, myocardial infarction, stroke), and death.14 This highly morbid condition can be treated with revascularization procedures. However, a substantial number of patients are not eligible for such interventions, and as a result, have extremely poor quality of life compared with those whose peripheral artery disease is not as advanced.15 Medical therapies that have been studied to improve limb salvage and survival include prostanoid administration and spinal cord stimulation; studies of the former yielded conflicting results, and studies of the latter yielded more promising results.1 Nevertheless, better treatment options have yet to be proven for these “no option” CLI patients. Gene therapy and cell therapy with bone marrow-derived progenitor cells are recent areas of research.7-11 The present study examined long-term outcomes from intramuscular administration of ABMNCs. AFS, the gold-standard end point for analyzing CLI treatment efficacy,1,16,17 is the primary end point in our study. We report a 5-year AFS of 74%, somewhat decreased from 86% at the 1-year time point.12 These percentages are compared with similar studies with limb salvage rates of 59% to 66.7% at <1 year18,19; another study using bone marrow cell therapy without isolating mononuclear cells reported AFS of 78% at 1 year.20 Our review of the literature did not find any studies that followed patient outcomes beyond 3 years. The study with 3-year outcomes was the Therapeutic Angiogenesis by Cell Transplantation trial that showed survival of 80% and 100% for patients with
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CLI due to atherosclerotic peripheral artery disease and Buerger disease, respectively; amputation-free rates were 60% and 91% for the same two etiologies, respectively.8 Even among CLI patients who had the option of undergoing revascularization procedures, 3-year limb salvage and AFS have been reported to be 73% and 31%, respectively.21 Additionally, in a recent study by Giles et al20 that examined AFS in CLI patients who received bone marrow cell therapy vs high-risk bypass surgery, they found that the rates of AFS at 1 year were similar at 78% and 69%, respectively. They noted that the cell therapy results met the Society of Vascular Surgery CLI objective performance goal of 71% AFS at 1 year.20,22 Interestingly, our results at 5 yearsdAFS of 74%dalso meet this goal. With most of the subjects available for follow-up, we were also able to determine that there were no known incidences of malignancy or clinically significant proliferative retinopathy at 5 years, although three patients had major cardiac events. The therapeutic mechanism of action of ABMNCs has been investigated in animal models23 and patients,24,25 where angiogenesis was seen in some of the treated subjects. The prevailing theory is that vascular growth occurs in the ischemic limb, perhaps via interaction of local vascular and inflammatory cells and the injected ABMNCs.26 However, the exact mechanism is still unclear and would be important to elucidate to facilitate future therapeutic endeavors. To identify any neovascularization occurring with ABMNC therapy in this study, four subjects initially had contrast arteriography and magnetic resonance angiography performed at baseline and at 12 weeks.12 However, these imaging techniques were abandoned, because they were limited by several factors, including venous contamination, elimination of patients with renal insufficiency, dependence on contrast delivery and image capture, and lack of resolution for arterioles. Therefore, an experimental positron emission tomographybased protocol with computed tomography using radiolabeled water was developed and used for these four subjects and reported in our initial 1-year results.12 Because of logistical difficulties with adequate follow-up of subjects, because they lived in different parts of the country and any vascular assessment done locally would be subject to variations in equipment, technician, and environment, imaging was not performed for the 5-year assessment. Although the long-term outcomes reported here are encouraging, we note some of the limitations of our study. For this initial investigation, we designed a phase I/II open-label study. There was no comparator group that could act as a control to see differences in the effects of ABMNC therapy. The relatively small number of participants in this study also precludes more definitive conclusions of safety and efficacy. However, the randomized, double-blind phase III clinical trial of 152 patients is currently under way, with the primary completion date estimated to be within the next year. The larger number of patients and the more rigorous design of this phase III study will better define the preliminary results from the current study. Furthermore, our study population is relatively healthier than the actual
CLI population; we excluded those with hemoglobin A1c >8.5%, renal insufficiency or failure, and left ventricular failure. These patients presumably also had significant financial means, because they were able to travel to our institution from various parts of the country, and therefore, likely have superior medical care; this selection bias indicates that the group of patients studied here is likely not representative of CLI patients as a whole. The phase III study will include a broader range of patients to reduce the “good risk” bias in the current study. CONCLUSIONS Long-term results of intramuscular injection of ABMNCs to “no option” CLI limbs show results that are comparable with other reports of patients who underwent surgical and endovascular interventions.16,27 Moreover, this mode of cellular therapy was shown to be safe in our phase I/II study. However, safety and effectiveness will need to be corroborated in the phase III MOBILE trial that recently completed enrollment. AUTHOR CONTRIBUTIONS Conception and design: MM Analysis and interpretation: TL, AJ, MW, MM Data collection: AJ, RM, PG, GA, AF, MM Writing the article: TL, MW Critical revision of the article: AJ, RM, PG, GA, AF, MM Final approval of the article: TL, AJ, RM, MW, PG, GA, AF, MM Statistical analysis: MW Obtained funding: MM Overall responsibility: MM REFERENCES 1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-society consensus for the management of peripheral arterial disease (TASC II). J Vasc Surg 2007;45(Suppl S):S5-67. 2. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:e463-654. 3. Kusumanto YH, van Weel V, Mulder NH, Smit AJ, van den Dungen JJ, Hooymans JM, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a doubleblind randomized trial. Hum Gene Ther 2006;17:683-91. 4. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114-23. 5. Powell RJ, Simons M, Mendelsohn FO, Daniel G, Henry TD, Koga M, et al. Results of a double-blind, placebo-controlled study to assess the
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safety of intramuscular injection of hepatocyte growth factor plasmid to improve limb perfusion in patients with critical limb ischemia. Circulation 2008;118:58-65. Shigematsu H, Yasuda K, Iwai T, Sasajima T, Ishimaru S, Ohashi Y, et al. Randomized, double-blind, placebo-controlled clinical trial of hepatocyte growth factor plasmid for critical limb ischemia. Gene Ther 2010;17:1152-61. Cobellis G, Silvestroni A, Lillo S, Sica G, Botti C, Maione C, et al. Long-term effects of repeated autologous transplantation of bone marrow cells in patients affected by peripheral arterial disease. Bone Marrow Transpl 2008;42:667-72. Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, et al. Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J 2008;156:1010-8. Barc P, Skóra J, Pupka A, Turkiewicz D, Dorobisz AT, Garcarek J, et al. Bone-marrow cells in therapy of critical limb ischaemia of lower extremities e own experience. Acta Angiologica 2006;12:155-66. Huang P, Li S, Han M, Xiao Z, Yang R, Han ZC. Autologous transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells improves critical limb ischemia in diabetes. Diabetes Care 2005;28:2155-60. Franz RW, Shah KJ, Johnson JD, Pin RH, Parks AM, Hankins T, et al. Short- to mid-term results using autologous bone-marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. Vasc Endovascular Surg 2011;45:398-406. Murphy MP, Lawson JH, Rapp BM, Dalsing MC, Klein J, Wilson MG, et al. Autologous bone marrow mononuclear cell therapy is safe and promotes amputation-free survival in patients with critical limb ischemia. J Vasc Surg 2011;53:1565-74.e1. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457-81. A prospective epidemiological survey of the natural history of chronic critical leg ischaemia. The I.C.A.I. Group (gruppo di studio dell’ischemia cronica critica degli arti inferiori). Eur J Vasc Endovasc Surg 1996;11:112-20. Sprengers RW, Teraa M, Moll FL, de Wit GA, van der Graaf Y, Verhaar MC. Quality of life in patients with no-option critical limb ischemia underlines the need for new effective treatment. J Vasc Surg 2010;52:843-9. 849.e1. Adam DJ, Beard JD, Cleveland T, Bell J, Bradbury AW, Forbes JF, et al. Bypass versus angioplasty in severe ischaemia of the leg (BASIL): multicentre, randomised controlled trial. Lancet 2005;366:1925-34.
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17. Benoit E, O’Donnell TF Jr, Iafrati MD, Asher E, Bandyk DF, Hallett JW, et al. The role of amputation as an outcome measure in cellular therapy for critical limb ischemia: implications for clinical trial design. J Transl Med 2011;9:165. 18. Amann B, Luedemann C, Ratei R, Schmidt-Lucke JA. Autologous bone marrow cell transplantation increases leg perfusion and reduces amputations in patients with advanced critical limb ischemia due to peripheral artery disease. Cell Transpl 2009;18:371-80. 19. Franz RW, Parks A, Shah KJ, Hankins T, Hartman JF, Wright ML. Use of autologous bone marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. J Vasc Surg 2009;50:1378-90. 20. Giles KA, Rzucidlo EM, Goodney PP, Walsh DB, Powell RJ. Bone marrow aspirate injection for treatment of critical limb ischemia with comparison to patients undergoing high-risk bypass grafts. J Vasc Surg 2015;61:134-7. 21. Engelhardt M, Boos J, Bruijnen H, Wohlgemuth W, Willy C, Tannheimer M, et al. Critical limb ischaemia: initial treatment and predictors of amputation-free survival. Eur J Vasc Endovasc Surg 2012;43:55-61. 22. Conte MS, Geraghty PJ, Bradbury AW, Hevelone ND, Lipsitz SR, Moneta GL, et al. Suggested objective performance goals and clinical trial design for evaluating catheter-based treatment of critical limb ischemia. J Vasc Surg 2009;50:1462-73. 1473.e1-3. 23. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, Duan J, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 2001;103:897-903. 24. Esato K, Hamano K, Li TS, Furutani A, Seyama A, Takenaka H, et al. Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease. Cell Transpl 2002;11:747-52. 25. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002;360:427-35. 26. van Weel V, van Tongeren RB, van Hinsbergh VW, van Bockel JH, Quax PH. Vascular growth in ischemic limbs: a review of mechanisms and possible therapeutic stimulation. Ann Vasc Surg 2008;22:582-97. 27. Jones DW, Schanzer A, Zhao Y, MacKenzie TA, Nolan BW, Conte MS, et al. Growing impact of restenosis on the surgical treatment of peripheral arterial disease. J Am Heart Assoc 2013;2: e000345.
Submitted Oct 8, 2015; accepted Jan 13, 2016.
Autologous bone marrow mononuclear cell therapy for critical limb ischemia is effective and durable Tiffany W. Liang, MD,a Andrea Jester, MD,a Raghu L. Motaganahalli, MD,b Michael G. Wilson, PhD,c Patricia G’Sell, RN,d George A. Akingba, MD, PhD,b Andres Fajardo, MD,b and Michael P. Murphy, MD,b Indianapolis, Ind Objective: We have previously shown that autologous bone marrow mononuclear cell (ABMNC) therapy improves measures of limb perfusion, rest pain, wound healing, and amputation-free survival (AFS) at 1 year in patients with critical limb ischemia (CLI). Long-term durability of ABMNC therapy for CLI remains unknown. The objective of the current study was to evaluate long-term clinical outcomes 5 years after treatment. Methods: Data were retrospectively gathered from a database and via a patient survey and review of medical records of patients previously enrolled in this phase I/II trial. AFS, freedom from major amputation, and freedom from major adverse limb events (MALE) were calculated using the product-limit estimate. The incidence of cardiac, malignant, and other medical events relevant to the safety of cell therapy were tabulated during the time from treatment to follow-up. Results: Twenty-one of the 24 patients (88%) who completed the initial 1-year phase I/II trial were available for the 5year analysis; AFS was 74% (95% confidence interval [CI], 0.53-0.87), freedom from major amputation was 78% (95% CI, 0.58-0.90), and freedom from MALE was 65% (95% CI, 0.45-0.80). Three patients (14%) had major cardiac events. There were no incidences of malignancies or diagnoses of clinically significant proliferative retinopathy. Fifteen patients (71%) report continued improvement in pain-free walking. Nineteen (90%) patients believed that the study was of significant medical value and would participate again. Conclusions: ABMNC therapy provides long-term freedom from AFS, major amputation, and MALE that are comparable with other reports of patients who underwent surgical and endovascular interventions for CLI. Furthermore, no patients developed tumorigenesis or clinically significant retinopathy. Because of the limited number of patients studied, our findings will need to be followed up in a larger phase III trial. (J Vasc Surg 2016;63:1541-5.)
Critical limb ischemia (CLI) is a debilitating condition characterized by rest pain, ulcers, or gangrene due to chronic ischemia.1 There is an estimated 500 to 1000 newly diagnosed cases of CLI per every 1 million people in Europe or North America.1 Current treatment includes
From the Department of Surgery,a Division of Vascular Surgeryb Division of Clinical Pharmacology,c and Indiana Center for Vascular Biology and Medicine,d Indiana University School of Medicine. Biomet Biologics, LLC (Warsaw, Ind) provided the MarrowStim cell isolation system for use in the initial study. They also provided the data for Fig 1. Otherwise, Biomet Biologics, LLC had no involvement in the study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. Clinical trial registration number NCT00113243. Author conflict of interest: none. Presented at the Thirty-fifth Annual Meeting of the Midwestern Vascular Surgical Society, Chicago, Ill, September 15-17, 2011. Correspondence: Michael P. Murphy, MD, Division of Vascular Surgery, Department of Surgery, Indiana University School of Medicine, 1801 Senate Blvd, MPC# 2-3500, Indianapolis, IN 46202 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2016 by the Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvs.2016.01.022
surgical or catheter-based revascularization procedures; medical and pharmacologic therapies can be used in “no option” patients (ie, if revascularization is not possible).1,2 Among these “no option” patients, approximately 40% will require a major amputation and up to 20% will have died within 6 months.1 Because of the high rate of morbidity and mortality for CLI patients who are unable to undergo successful reperfusion, there has been extensive research into alternative treatment strategies. These include gene therapy with vascular endothelial growth factor3,4 and hepatocyte growth factor5,6 as well as cell therapy with bone marrow-derived cells.7-11 The rationale behind these therapies is facilitation of angiogenesis in the ischemic limb. We have previously published 12-month results from an open-label, phase I/II clinical trial involving intramuscular injection of autologous bone marrow mononuclear cells (ABMNCs) into the affected limb, where we reported that this mode of therapy is safe and effective for increasing amputation-free survival (AFS),12 and on the basis of this information we have proceeded with a pivotal multicenter, randomized, double blinded phase III study, the MarrOwStim Peripheral Arterial Disease Kit for the Treatment of Critical LimB IschemIa in Subjects with Severe Peripheral ArteriaL DiseasE (MOBILE) trial. However, for cell therapy to be considered a viable option for longterm management, any beneficial effects should withstand 1541
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the test of time. Because there are few studies in the literature that show outcomes beyond 3 years after therapy, the objective of this report was to follow-up our previous study and determine if the initial promising results held up 5 years later. METHODS Patient selection. A complete description of initial patient selection and characteristics has been published previously.12 In brief, 29 patients (30 limbs) between the ages of 21 and 85 years with “no option” CLI from different parts of the United States were enrolled between September 2005 and March 2009. These patients had symptoms of rest pain and/or ulceration (21 limbs had rest pain only, six had rest pain and ulceration, and three had ulcers only) with an ankle brachial index #0.55 and/or toe brachial index #0.40, as well as an absence of a patent artery below the knee that was continuous to the foot, confirmed using contrast arteriography. An independent vascular surgeon had determined that bypass and/or endovascular approaches to improving perfusion were not possible options for management. As indicated previously, the institutional review board approved the trial protocol, and all patients provided written informed consent to participate in the study.12 Of the initially enrolled 29 patients, two patients died and three limbs were either lost to follow-up or had reached an end point by the 1-year time point. The remaining 24 patients (24 limbs) continued to be included in the study for long-term follow up. Study design. A detailed study design has been presented previously.12 In brief, this was a phase I/II openlabel, prospective study that consisted of a single-dose injection of ABMNCs isolated by using either conventional Ficoll density centrifugation (first 14 patients enrolled) or the MarrowStim (Biomet Biologics, Warsaw, Ind) pointof-care centrifugation system (15 patients, 16 limbs). The ABMNCs were injected intramuscularly to the affected limb distal to the anterior tibial tuberosity. The MarrowStim (Biomet Biologics) system is an alternative cell isolation method that maintains sterility and takes <20 minutes to perform, tested in this study to replace the traditional Ficoll system. These two isolation systems were found to yield comparable ABMNC recovery cell counts; as published previously, the average numbers of ABMNCs obtained after Ficoll and MarrowStim (Biomet Biologics) isolation were 1.3 6 0.7 109 and 2.0 6 1.6 109 cells, respectively (Fig 1).12 The initial study subjects that remained continued to be followed to examine long-term outcomes at 5 years. To this end, we sent out questionnaires, conducted telephone interviews, and retrospectively reviewed their medical records. Outcome measures. The primary outcome was AFS, defined as no major amputation and no death. Secondary outcomes were freedom from major amputation and
Fig 1. Comparison of autologous bone marrow mononuclear cell (ABMNC) recovery from Ficoll and MarrowStim (Biomet Biologics, Warsaw, Ind) isolation systems. Data from Biomet Biologics, LLC.
Table. Censoring mechanisms for three time-to-event outcomes Outcomes AFS Freedom from major amputation Freedom from MALE
Events Death Amputation Amputation Amputation MALE
Censoring mechanisms Lost to follow-up Study termination Death Lost to follow-up Study termination Death Lost to follow-up Study termination
AFS, Amputation-free survival; MALE, major adverse limb events.
freedom from major adverse limb events (MALE). Major amputation was defined as any amputation proximal to the malleolar level, such as a below-knee or above-knee amputation. MALE was defined as any major amputation, minor amputation including transmetatarsal amputation, or surgical reintervention (eg, angioplasty, stenting, or bypass). However, because all subjects were classified in the “no option” designation, none received surgical intervention after study initiation. Therefore, MALE was effectively any major or minor amputation. Additionally, to examine long-term safety, we tabulated the incidence of major adverse cardiac events, malignancies, and clinically significant proliferative retinopathy that might have occurred from the time of ABMNC injection to follow-up. Major adverse cardiac events were defined as Q-wave myocardial infarction, stroke, or death. Malignancy was defined as biopsy-proven malignant transformation of a lesion. Statistical analysis. A summary of events and censoring mechanisms for each outcome is provided in the Table. The product-limit estimates13 for each outcome and the associated 95% confidence intervals (CIs) were calculated. The upper and lower bounds of the 95% pointwise CI only exist at clinical failures and are undefined for censored observations. Using these product-limit estimates, the survival curves for each of the three outcomes were plotted. Under each plot,
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Fig 2. Kaplan-Meier curve of 5-year amputation-free survival (AFS).
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Fig 4. Kaplan-Meier curve of 5-year freedom from major adverse limb events (MALE).
experienced major cardiac events. None reported malignancies or diagnoses of clinically significant proliferative retinopathy. Other than adverse events, 15 patients (71%) reported continued improvement in pain-free walking on the basis of a walking impairment questionnaire. None reported deterioration of walking ability. When asked the question, 19 patients (91%) believed that the study was of significant medical value and would participate again. DISCUSSION
Fig 3. Kaplan-Meier curve of 5-year freedom from major amputation.
the number of limbs entering the interval at risk is annotated under the label of the major tick mark. RESULTS Consolidating previous knowledge of subjects from the initial 1-year study and new information gathered from the questionnaires and telephone interviews at 5 years, we compiled the results. Twenty-one of the 24 patients (88%) who did not die or drop out of the 1-year phase I/II trial responded to follow-up. Of the original 29 subjects (30 limbs), two patients had died (one from suicide), four limbs had minor amputations, two limbs had major amputations, and three limbs were either lost to followup or had reached an end point by the end of the 1-year study. By the 5-year time point, three additional limbs underwent major amputations. At 5 years, AFS was 74% (95% CI, 0.53-0.87; Fig 2); freedom from major amputation was 78% (95% CI, 0.58-0.90; Fig 3); and freedom from MALE was 65% (95% CI, 0.45-0.80; Fig 4). Regarding the 5-year incidence of adverse events, three of the 21 subjects who had follow-up at 5 years (14%)
The natural history of CLI frequently involves need for amputation, major cardiovascular events (eg, myocardial infarction, stroke), and death.14 This highly morbid condition can be treated with revascularization procedures. However, a substantial number of patients are not eligible for such interventions, and as a result, have extremely poor quality of life compared with those whose peripheral artery disease is not as advanced.15 Medical therapies that have been studied to improve limb salvage and survival include prostanoid administration and spinal cord stimulation; studies of the former yielded conflicting results, and studies of the latter yielded more promising results.1 Nevertheless, better treatment options have yet to be proven for these “no option” CLI patients. Gene therapy and cell therapy with bone marrow-derived progenitor cells are recent areas of research.7-11 The present study examined long-term outcomes from intramuscular administration of ABMNCs. AFS, the gold-standard end point for analyzing CLI treatment efficacy,1,16,17 is the primary end point in our study. We report a 5-year AFS of 74%, somewhat decreased from 86% at the 1-year time point.12 These percentages are compared with similar studies with limb salvage rates of 59% to 66.7% at <1 year18,19; another study using bone marrow cell therapy without isolating mononuclear cells reported AFS of 78% at 1 year.20 Our review of the literature did not find any studies that followed patient outcomes beyond 3 years. The study with 3-year outcomes was the Therapeutic Angiogenesis by Cell Transplantation trial that showed survival of 80% and 100% for patients with
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CLI due to atherosclerotic peripheral artery disease and Buerger disease, respectively; amputation-free rates were 60% and 91% for the same two etiologies, respectively.8 Even among CLI patients who had the option of undergoing revascularization procedures, 3-year limb salvage and AFS have been reported to be 73% and 31%, respectively.21 Additionally, in a recent study by Giles et al20 that examined AFS in CLI patients who received bone marrow cell therapy vs high-risk bypass surgery, they found that the rates of AFS at 1 year were similar at 78% and 69%, respectively. They noted that the cell therapy results met the Society of Vascular Surgery CLI objective performance goal of 71% AFS at 1 year.20,22 Interestingly, our results at 5 yearsdAFS of 74%dalso meet this goal. With most of the subjects available for follow-up, we were also able to determine that there were no known incidences of malignancy or clinically significant proliferative retinopathy at 5 years, although three patients had major cardiac events. The therapeutic mechanism of action of ABMNCs has been investigated in animal models23 and patients,24,25 where angiogenesis was seen in some of the treated subjects. The prevailing theory is that vascular growth occurs in the ischemic limb, perhaps via interaction of local vascular and inflammatory cells and the injected ABMNCs.26 However, the exact mechanism is still unclear and would be important to elucidate to facilitate future therapeutic endeavors. To identify any neovascularization occurring with ABMNC therapy in this study, four subjects initially had contrast arteriography and magnetic resonance angiography performed at baseline and at 12 weeks.12 However, these imaging techniques were abandoned, because they were limited by several factors, including venous contamination, elimination of patients with renal insufficiency, dependence on contrast delivery and image capture, and lack of resolution for arterioles. Therefore, an experimental positron emission tomographybased protocol with computed tomography using radiolabeled water was developed and used for these four subjects and reported in our initial 1-year results.12 Because of logistical difficulties with adequate follow-up of subjects, because they lived in different parts of the country and any vascular assessment done locally would be subject to variations in equipment, technician, and environment, imaging was not performed for the 5-year assessment. Although the long-term outcomes reported here are encouraging, we note some of the limitations of our study. For this initial investigation, we designed a phase I/II open-label study. There was no comparator group that could act as a control to see differences in the effects of ABMNC therapy. The relatively small number of participants in this study also precludes more definitive conclusions of safety and efficacy. However, the randomized, double-blind phase III clinical trial of 152 patients is currently under way, with the primary completion date estimated to be within the next year. The larger number of patients and the more rigorous design of this phase III study will better define the preliminary results from the current study. Furthermore, our study population is relatively healthier than the actual
CLI population; we excluded those with hemoglobin A1c >8.5%, renal insufficiency or failure, and left ventricular failure. These patients presumably also had significant financial means, because they were able to travel to our institution from various parts of the country, and therefore, likely have superior medical care; this selection bias indicates that the group of patients studied here is likely not representative of CLI patients as a whole. The phase III study will include a broader range of patients to reduce the “good risk” bias in the current study. CONCLUSIONS Long-term results of intramuscular injection of ABMNCs to “no option” CLI limbs show results that are comparable with other reports of patients who underwent surgical and endovascular interventions.16,27 Moreover, this mode of cellular therapy was shown to be safe in our phase I/II study. However, safety and effectiveness will need to be corroborated in the phase III MOBILE trial that recently completed enrollment. AUTHOR CONTRIBUTIONS Conception and design: MM Analysis and interpretation: TL, AJ, MW, MM Data collection: AJ, RM, PG, GA, AF, MM Writing the article: TL, MW Critical revision of the article: AJ, RM, PG, GA, AF, MM Final approval of the article: TL, AJ, RM, MW, PG, GA, AF, MM Statistical analysis: MW Obtained funding: MM Overall responsibility: MM REFERENCES 1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-society consensus for the management of peripheral arterial disease (TASC II). J Vasc Surg 2007;45(Suppl S):S5-67. 2. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 2006;113:e463-654. 3. Kusumanto YH, van Weel V, Mulder NH, Smit AJ, van den Dungen JJ, Hooymans JM, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a doubleblind randomized trial. Hum Gene Ther 2006;17:683-91. 4. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114-23. 5. Powell RJ, Simons M, Mendelsohn FO, Daniel G, Henry TD, Koga M, et al. Results of a double-blind, placebo-controlled study to assess the
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Submitted Oct 8, 2015; accepted Jan 13, 2016.