Thrombotic Microangiopathy In Metastatic Melanoma Patients Treated with Adoptive Cell Therapy and Total Body Irradiation

Introduction

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.(1) 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 prior to T-cell transfer in a mouse model correlated with a more favorable dose response relationship.(2) 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 total body irradiation (TBI) (2 Gy or 12 Gy), objective response rates using RECIST criteria were 49%, 52%, and 72% respectively. Complete response (CR) rates in these three consecutive trials were 12%, 20%, and 40% respectively. These findings strongly suggested that the addition of high-dose TBI with autologous stem cell supportto cyclophosphamide and fludarabine pre-conditioning improved CR rates to ACT.(3,4) Based on these preliminary data and the observation that nearly all complete responses remained durable over 5-year follow-up, aprospective randomized trial was initiated to evaluate whether the addition of 12Gy TBI to TIL treatment leads to greater complete response 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 non-randomized experience, thrombotic microangioapathy (TMA) was a notable complication that developed in a subset of patients that 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, total body irradiation, immunosuppressant medications (i.e. calcineurin inhibitors), GVHD, and various infections. Establishing a diagnosis of TMA in SCT patients can be challenging, as hematologic and renal abnormalities are frequently present for many reasons. Moreover, identifying the precise causative agent of TMA is difficult as 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, as other confounding influences are absent. This paper describes the incidence, presentation and course of radiation-associated TMA.

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 total body irradiation (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 (12Gy). Eligibility criteria included age of 18 years or older, measurable metastatic melanoma and no contraindications to high-dose aldesleukin or administration of total body irradiation. The basic chemotherapy regimen consisted of 60 mg/kg cyclophosphamide daily for two days and 25 mg/m² fludarabine daily for five 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 six hours apart over three 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 six hours apart. All patients were treated with a linear accelerator using energies higher than 4MV. One day after T-cell administration, patients received at least 3×10⁶/kg 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 3-4 months and every 2-3 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 thrombotic microangiopathy were collected well after the expected time frame had elapsed for all acute side effects of aldesleukin or autologous tumor infiltrating lymphocytes infusion.

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.

Statistical methods

The median values were obtained for characteristics of patients in the two trials. The Kaplan-Meier method was employed to calculate cumulative incidence. A two-sided Fisher's exact test was used to compare cancer outcomes between patients who did and did not develop TMA.

Results

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.

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 to 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 post-treatment (Figure 1). The median follow-up interval for all patients was 24 months (6 to 82 months). Patients had very few comorbidities prior to treatment; four patients were previously diagnosed with mild essential hypertension, well controlled on a single antihypertensive agent and one patient had hypothyroidism.

Given the uncertainty of complications associated with new protocols, the diagnosis of TMA was established by renal biopsy in three patients in the pilot trial. Renal biopsies all showed histologic features consistent with TMA (Figure 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 out 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) (Figure 2). New-onset hypertension was a feature in 13 out of 15 (87%) of the patients who developed TMA. Proteinuria was present in all patients, though most had subnephrotic proteinuria. Mean urine protein/creatinine ratio at time of diagnosis, was 1.8 (range 0.4-3.6; ULN 0.16 mg/mg). Hemoglobinuria (14 out of 15 patients, 93%) and red blood cells were additional findings on urinalysis. ADAMTS13 levels were measured in 3 patients and all were normal.

New-onset anemia was observed in 12 out 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 out of 15 patients (93%) but only two required any platelet transfusions and there were no bleeding sequelae. Low haptoglobin (11 out of 15 patients, 73%) was also frequently seen at diagnosis. Elevations of lactate dehydrogenase values were also encountered, but confounded in our study by pre-existing 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.

Discussion

TMA is a known late side effect after high-dose myeloablative chemotherapy and total body irradiation (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.(7) The usual time of onset for 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.(11)

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 bone marrow transplantation (BMT) population.(8) 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.(12) 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) As 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 anti-tumor 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 three times daily over 3 days) with renal shielding and 26% with 14 Gy radiation.(20) 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. Additionally, increased fractionation and low dose rates (<10 cGy/min) are associated with lower incidence of toxicity.(8) 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.(21) 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.(22) As 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 trialsare 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 anti-melanoma 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.

Conclusion

In a patient population receiving cyclophosphamide and fludarabine with TBI prior to autologous T-cell therapy for melanoma, thrombotic microangiopathy (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 anti-hypertensive 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 five 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.