Thrombotic microangiopathy occurs in a significant subset of patients following total body irradiation preconditioning for adoptive cell therapy and can be successfully managed with supportive care.
Thrombotic microangiopathy (TMA) is a complication that developed in some patients receiving 12 Gy total body irradiation (TBI) in addition to lymphodepleting preparative chemotherapy prior to infusion of autologous tumor-infiltrating lymphocytes (TIL) with high-dose aldesleukin (IL-2). This article describes the incidence, presentation, and course of radiation-associated TMA.
The data for patients with metastatic melanoma who received ACT with TIL plus aldesleukin following myeloablative chemotherapy and 12-Gy TBI was examined, in order to look at patient characteristics and the natural history of TMA.
The median time to presentation was approximately 8 months after completing TBI. The estimated cumulative incidence of TMA was 31.2% (median follow-up of 24 months). Noninvasive criteria for diagnosis included newly elevated creatinine levels, new-onset hypertension, new-onset anemia, microscopic hematuria, thrombocytopenia, low haptoglobin, and elevated lactate dehydrogenase values. Once diagnosed, patients were managed with control of their hypertension with multiple agents and supportive red blood cell transfusions. TMA typically stabilized or improved and no patient progressed to dialysis. TMA was associated with a higher probability of an antitumor response.
TMA occurs in approximately a third of patients treated with a lymphodepleting preparative chemotherapy regimen with TBI prior to autologous T cell therapy. The disease has a variable natural history, however, no patient developed end-stage renal failure. Successful management with supportive care and aggressive hypertension control is vital to the safe application of a systemic therapy that has shown curative potential for patients with disseminated melanoma. Cancer 2014;120:1426–1432. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Adoptive cell therapy (ACT) using ex vivo expanded autologous tumor-infiltrating lymphocytes (TIL) can mediate the regression of bulky metastatic melanoma when administered along with high-dose aldesleukin (IL-2) following a lymphodepleting preparative regimen consisting of cyclophosphamide and fludarabine. The lymphocytes infiltrating human melanoma metastases contain populations with autologous tumor recognition in the vast majority of patients. In studies on improving ACT, escalating doses of total body irradiation (TBI) prior to T cell transfer in a mouse model correlated with a more favorable dose response relationship. Thus, increased lymphodepletion with the addition of TBI was thought to enhance tumor treatment efficacy, prompting further human trials. In a series of consecutive trials using a cyclophosphamide-fludarabine preparative chemotherapy regimen alone or in combination with TBI (2 Gy or 12 Gy), objective response rates using RECIST criteria were 49%, 52%, and 72%, respectively. Complete response (CR) rates in these 3 consecutive trials were 12%, 20%, and 40%, respectively. These findings strongly suggest that the addition of high-dose TBI with autologous stem cell support to cyclophosphamide and fludarabine preconditioning improved CR rates to ACT.[3, 4] Based on these preliminary data and the observation that nearly all CRs remained durable over 5 years follow-up, a prospective randomized trial was initiated to evaluate whether the addition of 12-Gy TBI to TIL treatment leads to greater CR rates and overall survival compared to patients not receiving TBI. Along with assessment of efficacy, the toxicity associated with these regimens was evaluated. In the prior nonrandomized experience, thrombotic microangiopathy (TMA) was a notable complication that developed in a subset of patients who received 12 Gy TBI, but not those receiving just chemotherapy or chemotherapy plus 2 Gy TBI.
TMA is a well-described complication after allogeneic stem cell transplantation (SCT) and is attributed to factors including high-dose chemotherapy, TBI, immunosuppressant medications (ie, calcineurin inhibitors), graft-versus-host disease, and various infections. Establishing a diagnosis of TMA in SCT patients can be challenging, because hematologic and renal abnormalities are frequently present for many reasons. Moreover, identifying the precise causative agent of TMA is difficult because patients are exposed to multiple risk factors, often simultaneously. Unlike with SCT, our protocols using autologous T cells and TBI provide a unique opportunity to directly examine the role of radiation in development of TMA, because other confounding influences are absent. This article describes the incidence, presentation, and course of radiation-associated TMA.
MATERIALS AND METHODS
Two clinical trials were analyzed from the National Cancer Institute (NCI), part of the National Institutes of Health (NIH) in Bethesda, Maryland. In the initial pilot trial, patients with metastatic melanoma received ACT with TIL plus aldesleukin following myeloablative chemotherapy and 12-Gy TBI (always with autologous stem cell support). Twenty-five patients were enrolled between April 2006 and August 2007. The ongoing randomized control trial (RCT), which began accruing in July 2011, randomizes patients to preparative chemotherapy with or without TBI (12 Gy). Eligibility criteria included age of 18 years or older, measurable metastatic melanoma, and no contraindications to high-dose aldesleukin or administration of TBI. The basic chemotherapy regimen consisted of 60 mg/kg cyclophosphamide daily for 2 days and 25 mg/m2 fludarabine daily for 5 days prior to T cell transfer and aldesleukin administration.
For patients from the initial trial and also those randomized to the TBI arm, the dose was 12 Gy delivered in 2-Gy fractions twice daily, at least 6 hours apart over 3 days (Table 1). TBI was delivered with lateral fields using extended SSD/SAD values of 200 to 500 cm (depending on machine/vault size) and partial transmission lung blocks. Patients then received AP/PA mediastinal boost fields using full blocks in the lung area. The mediastinal boost fields were delivered at a dose of 100 cGy per fraction for a total dose of 600 cGy, also at least 6 hours apart. All patients were treated with a linear accelerator using energies higher than 4 MV. One day after T cell administration, patients received at least 3 × 106/kg granulocyte colony-stimulating factor (G-CSF)–mobilized autologous CD34+ hematopoietic stem cells collected and cryopreserved previously. Patients were discharged when their absolute neutrophil counts were > 1000/mm3 for 3 consecutive days, their hemoglobin was stable at > 8 g/dL, and their platelet counts were stable at > 30,000/mm3. They were evaluated at least every month for 3 to 4 months and every 2 to 3 months for a year. Thereafter, patients continued to be seen at the NIH biannually or annually while they had a cancer response; those with new melanoma disease were referred back to their home oncologist for other management, truncating the length of formal follow-up. All data presented here in the analysis of TMA were collected well after the expected time frame had elapsed for all acute side effects of aldesleukin or autologous TIL infusion.
Table 1. 12-Gy Total Body Irradiation Treatment Algorithm
Dose of 720,000 IU/kg every 8 hours as tolerated to a maximum of 15 doses.
Noninvasive diagnostic criteria for TMA included acute hemolytic anemia, elevated lactate dehydrogenase (or a rise above previous values), low haptoglobin, thrombocytopenia, and acute kidney injury (acute rise in creatinine above previously established baseline). Renal biopsy was not required for diagnosis but was included if available. Patients were considered to be hypertensive if diastolic blood pressures were ≥ 90 mm Hg and systolic blood pressures were ≥ 140 mm Hg consistently for at least 3 measurements.
The median values were obtained for characteristics of patients in the 2 trials. The Kaplan-Meier method was employed to calculate cumulative incidence. A 2-sided Fisher's exact test was used to compare cancer outcomes between patients who did and did not develop TMA.
Twenty-five patients were enrolled in the pilot single-arm study using 12 Gy TBI between April 2006 and August 2007 (Trial 1). Eighty-six patients were enrolled in the ongoing RCT between July 2011 and May 2013. The patients' characteristics are listed in Table 2.
Table 2. Baseline Patient Characteristics
Trial 2, Randomized to TBI
Abbreviation: TBI, total body irradiation.
No. of patients
Median age (y)
Median follow-up (mo)
Baseline serum creatinine (mg/dL)
Patients receiving plasma exchange
Of the 111 patients enrolled on both trials, 67 patients received TBI. Of patients exposed to TBI, 15 (22%) were diagnosed with thrombotic microangiopathy. Only patients receiving TBI developed TMA. Median age of the patients who developed TMA was 52 years (range, 30-64 years) and included 7 men and 8 women (Table 3). The median time to presentation was approximately 8 months after completing TBI, with a range of 4 to 12 months. The estimated cumulative incidence of TMA by the Kaplan-Meier method was 31.2%, with no new cases presenting beyond 12 months after treatment (Fig. 1). The median follow-up interval for all patients was 24 months (range, 6-82 months). Patients had very few comorbidities prior to treatment: 4 patients were previously diagnosed with mild essential hypertension, which was well controlled on a single antihypertensive agent, and 1 patient had hypothyroidism.
Table 3. Characteristics of Patients With Thrombotic Microangiopathy (n = 15)
At date of total body irradiation (TBI) with tumor-infiltrating lymphocyte (TIL) treatment.
From date of TBI with TIL treatment to May 2013 or date of death.
Given the uncertainty of complications associated with new protocols, the diagnosis of TMA was established by renal biopsy in 3 patients in the pilot trial. Renal biopsies all showed histologic features consistent with TMA (Fig. 2). Light microscopy showed arteriolar and/or glomerular intracapillary thrombosis, often with accumulation of fragmented erythrocytes within capillary lumens. Glomeruli showed global widening of subendothelial spaces with double contours of the basement membranes. There were varying degrees of mesangiolysis. Electron microscopy showed electron lucent material in widened subendothelial spaces, fibrin in some capillaries, and red blood cell fragmentation. No electron-dense deposits were identified.
After establishing the histologic diagnosis of TMA, subsequent cases of TMA were identified using noninvasive criteria as defined in Table 4. All 15 of 15 patients (100%) who developed TMA had newly increased creatinine values. Peak creatinine values ranged from 1.23 to 3.70 mg/dL (normal reference range, 0.77-1.19 mg/dL) (Fig. 3). New-onset hypertension was a feature in 13 of 15 (87%) of the patients who developed TMA. Proteinuria was present in all patients, although most had subnephrotic proteinuria. Mean urine protein/creatinine ratio at time of diagnosis, was 1.8 (range 0.4-3.6; upper limit of the normal [ULN] range, 0.16 mg/mg). Hemoglobinuria (14 of 15 patients, 93%) and red blood cells were additional findings on urinalysis. ADAMTS13 levels were measured in 3 patients and all were normal.
Table 4. Clinical Markers for Diagnosis of Thrombotic Microangiopathy
Abbreviations: HTN, hypertension; LDH, lactate dehydrogenase; TBI, total body irradiation; TMA, thrombotic microangiopathy.
TBI without TMA
TBI with TMA
New-onset anemia was observed in 12 of 15 patients (80%). Five patients did not require any red blood cell transfusion for their TMA. The other 10 patients received between 3 and 30 units of PRBC (median, 6 units) over a median interval of 2 months (maximum interval of 10 months) before stabilizing or recovering. New thrombocytopenia occurred in 14 of 15 patients (93%) but only 2 required any platelet transfusions and there were no bleeding sequelae. Low haptoglobin (11 of 15 patients, 73%) was also frequently seen at diagnosis. Elevations of lactate dehydrogenase values were also encountered, but confounded in our study by preexisting elevations common in patients with metastatic melanoma. Schistocytes were a feature at some time on peripheral blood smears of all patients with TMA. Reticulocyte counts were normal in 10 patients and high in 5 patients.
Two patients from the original trial underwent plasmapheresis, but this treatment did not appear to alter the course of thrombotic microangiopathy or hematologic parameters. Once TMA was diagnosed, patients were followed closely by physicians at and outside the NIH for frequent monitoring of hematologic laboratory values. Transfusions took place at the NIH and also outside the NIH as patients sometimes received supportive transfusions under their local physicians' supervision. Resolution of the anemia typically coincided with improvement in serum creatinine values. Some patients also received epoetin alfa injections or iron supplementation. No patients received steroids.
The new-onset hypertension was frequently refractory to single agent, first-line antihypertensives. Typical combinations needed included an angiotensin-converting enzyme (ACE) inhibitor (lisinopril) or an angiotensin receptor blocker (losartan), beta-blocker (metoprolol or labetalol), and 1 or 2 diuretics (hydrochlorothiazide, furosemide, acetazolamide, and/or spironolactone). Occasionally, a calcium channel blocker (amlodipine) or an α2 adrenergic agonist (clonidine) were used for refractory hypertension. Patients regularly required 3 or 4 agents for adequate blood pressure control.
Three of the patients had normalization of their creatinine values (Table 5) and were able to be taken off of antihypertensive medication 3 to 6 months after diagnosis of TMA. No patients required dialysis. Of the 12 other patients, 10 continue on their hypertension management regimen and 2 died from progression of their metastatic melanoma. The only other significant complications in these patients include one patient who developed transient hypertensive retinopathy on presentation with TMA and one other patient who has developed red cell aplasia, likely unrelated to her TMA diagnosis. All other patients in these protocols were screened retrospectively and no clear evidence of subclinical TMA was encountered.
Normal creatinine (Cr) reference range, 0.77-1.19 mg/dL.
Examining cancer treatment outcomes, 14 of 15 patients (93%) were partial or complete responders to ACT. Three of these responders subsequently progressed at 10, 19, and 22 months after treatment. Nine of the remaining 11 patients are currently maintaining ongoing responses, with the other 2 patients dying from unrelated causes (normal pressure hydrocephalus with dementia and Merkel cell carcinoma).
A 2-tailed Fisher's exact test of cancer response rates comparing patients who received TBI but did not develop TMA (response rate [RR] = 46%) with those patients who received TBI and did develop TMA (RR = 93%) showed a significant difference (P2 value = .0017; Table 6). This association between TMA and a higher response rate was also seen when patients with TMA were compared to the other group of TMA-free patients who had not received TBI (RR = 45%; P2 value = .0029; Table 6). These analyses of response rates were confined to patients who had at least 12 months of documented creatinine values before assigning them to a TMA status to avoid bias against detection of TMA in nonresponders with shorter durations of follow-up. Including all patients in the analysis, regardless of follow-up, resulted in P values of .0004 and .0006, respectively (data not shown).
Table 6. Cancer Outcomes: Patients With No TBI vs TBI With TMA vs TBI Without TMA
Response Rate (%)
Fisher's Exact Test
Abbreviations: CR, complete responder; NR, nonresponder; PR, partial responder; TBI, total body irradiation; TMA, thrombotic microangiopathy.
P of 2-tailed Fisher's exact test comparing patients who never received TBI with those patients who did receive TBI and developed TMA.
P of 2-tailed Fisher's exact test comparing patients who received TBI but who did not develop TMA with those patients who did receive TBI and developed TMA.
TMA is a known late side effect after high-dose myeloablative chemotherapy and TBI. Clinical signs of this late-onset renal dysfunction include hypertension, disproportionately severe anemia, edema, proteinuria, hematuria, elevated serum creatinine, and decreased glomerular filtration rate.[5, 6] TBI has also been considered a predisposing factor for hemolytic uremic syndrome which can display a presentation similar to radiation nephritis. The usual time of onset for bone marrow transplantation (BMT)-associated nephropathy is 8 to 12 months after transplantation, with 80% of events occurring during the first year.[8-10] In patients developing TMA in the setting of allotransplantation, a subset of these BMT patients eventually required dialysis though no formal statistics are available.
TMA is thought to be secondary to endothelial injury caused by radiation therapy given after chemotherapy preconditioning and has previously been reported primarily in the BMT population. Radiation-related renal injury occurs from degeneration and sclerosis of arterioles and capillaries, narrowing or occlusion of the lumina, and secondary degeneration of glomeruli and tubules associated with interstitial fibrosis. Fibrin thrombi are found within glomeruli and involve arterioles and interlobular arteries. Light microscopy of renal biopsies show irregularity of capillary loop outlines, mesangiolysis and mesangial hypercellularity.[13, 14] Because tumor responses appear to be associated with the occurrence of TMA, there is the possibility that this TMA is related to an autoimmune phenomenon destroying ADAMTS13 as in some forms of TTP. ADAMTS13 was measured in three of the most recently diagnosed TMA patients, and low levels were not encountered. Nevertheless it remains possible that a cytokine or immune cell mediated contribution from the antitumor T cell therapy could be contributing to the incidence of TMA seen here.
The radiation dose associated with a 5% risk of renal dysfunction at 5 years is reported to be 14 Gy in adults and less than 12 Gy in children after TBI.[15-19] Lawton et al reported a 18-month risk of late renal dysfunction of 6% for 12 Gy (fractionated 3 times daily over 3 days) with renal shielding and 26% with 14 Gy radiation. Similar data was confirmed by the study from Miralbell et al who noted that the TBI dose–related kidney dysfunction risk at 18 months was 5%, 26% and 45% for those patients receiving 10 Gy, 12 Gy, and 13.5 Gy TBI respectively, showing there is a dose-response relationship between the dose of TBI and the frequency of chronic renal failure. In addition, increased fractionation and low dose rates (< 10 cGy/minute) are associated with lower incidence of toxicity. It is notable that in a trial using lower doses of TBI, no cases of TMA developed.
Cytotoxic chemotherapy potentiates the effects of radiation on the kidneys. Patients receiving fludarabine as part of their preparative regimen have a significantly increased risk of chronic kidney disease. Fludarabine may contribute to inhibition of repair of radiation-induced chromosome breaks and cell repopulation, cell synchronization to a more radiosensitive cell cycle phase, and S-phase cell loss by apoptosis. Because the risk of TMA is lower in comparable allogeneic stem cell transplant patients receiving TBI and identically dosed cyclophosphamide, fludarabine may be additive in renal toxicity and contribute to the higher incidence of TMA in post-ACT patients. Early, reversible renal failure directly related to chemotherapy does not appear related to late radiation-related nephropathy.
Angiotensin-converting enzyme inhibitors (AEIs) and angiotensin II type-1 receptor blockers (ARBs) may be of use in mitigating radiation-induced renal injury to target this pathway. An initial study showed that prophylactic usage of captopril and losartan were equally effective as mitigators of radiation nephropathy in an animal model and further trials are pending.[23-26] These preliminary data have led to the usage of an ACE inhibitor or angiotensin receptor blocker as the primary initial agent in management of hypertension in patients with TMA.
Because the consequences and durations of TMA seen in these patients have been limited and no patient progressed to end-stage renal failure even with follow-up beyond 5 years, it has not excluded our evaluation of TBI as part of the preparation for T cell transfer. In addition, the unexplained association between antimelanoma responses and TMI may have a favorable impact on the risk-benefit ratio for the patients undergoing TBI. Therefore, the decision to use TBI prior to adoptive cell therapy will depend primarily on whether or not the randomized trial reveals substantial benefits in efficacy for patients dealing with a lethal metastatic cancer.
In a patient population receiving cyclophosphamide and fludarabine with TBI prior to autologous T cell therapy for melanoma, TMA occurred 4 to 12 months after treatment in almost a third of patients. Once diagnosed, these patients were managed with supportive care and aggressive hypertension control with ACE inhibitors as the cornerstone to the antihypertensive regimen. The natural history of the disease process varied, with some patients recovering their renal function, with reversal of anemia and hypertension over an interval of several months. Other patients, however, experienced recovery of their kidney dysfunction but continued to need long-term management of hypertension. Even in the most severe cases, no patients proceeded to dialysis, in some cases with follow-up beyond 5 years. The recognition and successful management of this complication will be vital to the safe application of a systemic therapy that has shown curative potential for patients with disseminated melanoma.
All financial and material support for the research and the work was provided by the National Institutes of Health.