Cisplatin-induced haemolytic uraemic syndrome associated with a novel intronic mutation of CD46 treated with eculizumab
A previously well 27-month-old boy with no family history of kidney disease presented with a cervical mass unresponsive to antibiotics. The plasma lactate dehydrogenase (LDH) was elevated at 1600 iu/L (normal 266–500). Biopsy of the mass confirmed neuroblastoma with MYCN gene amplification. Bone marrow examination and metaiodobenzylguanidine (MIBG) scan showed no evidence of distant metastases. Pre-treatment, the plasma creatinine concentration was 21 μmol/L, and the glomerular filtration rate (GFR; measured by plasma disappearance of Technetium (Tc)-99m labelled Diethylene Triamine Pentacaetic Acid (DTPA) was 107 mL/min/1.73 m2 body surface area. Therapy was started with a combination of vincristine, etoposide, carboplatin, cisplatin and cyclophosphamide.\nOn Day 45 of treatment (35 days after the first cisplatin dose, 15 days after the second), the GFR had fallen to 60 mL/min/1.73 m2. This was presumed to be due to acute cisplatin nephrotoxicity. Two days later, the patient became fluid overloaded and hypertensive. He was profoundly pancytopaenic, which was attributed to chemotherapy-induced myelosuppression. The hypertension became increasingly severe (maximum value recorded 160 mmHg systolic) and he required five agents to control the blood pressure.\nHe required seven infusions of platelets and four of red blood cells between Days 50 and 59, suggesting that the pancytopaenia was not solely due to myelosuppression and the plasma creatinine concentration rose from 39 to 140 μmol/L. A diagnosis of HUS was considered. A blood film confirmed the presence of schistocytes, the LDH had risen to 2438 iu/L and the plasma haptoglobin concentration was <0.06 g/L (normal 0.5–2.0 g/L). Serum complement C3 (1.06 g/L) and C4 (0.20 g/L) concentrations were normal. Renal biopsy was considered unsafe at this time, but the results above were considered compatible with a diagnosis of HUS, likely due to cisplatin. Stool and blood cultures were negative, as were serological tests for HIV1&2. ADAMTS13 (von Willebrand factor-cleaving protease) levels were 80% of control values.\nDespite the dearth of information concerning the pathogenesis of cisplatin/carboplatin-associated chemotherapy, we opted for therapy with eculizumab partly because it was felt that his severe hypertension, profound thrombocytopaenia and small size made plasma exchange hazardous. He received the first dose of 600 mg the following day when his plasma creatinine was 155 μmol/L. The next day the plasma creatinine peaked at 160 μmol/L and it steadily decreased thereafter (). He also required progressively less blood product support. After a further dose of 300 mg a week later, he was maintained on fortnightly doses of 300 mg according to the manufacturer's dosing guidelines. Five weeks after administration of the first dose of eculizumab, the plasma creatinine concentration had reduced to 34 μmol/L, the plasma LDH was 532 iu/L and the GFR was 67 mL/min/1.73 m2. A decision was made to avoid further exposure to platinum-containing agents, and therapy was changed to a combination of vincristine, topotecan and doxorubicin. There was little further change in tumour size and, 126 days after starting chemotherapy, the tumour was surgically removed with clear excision margins and eculizumab was discontinued. At this time, the platelet count was 181 × 109/L and the plasma creatinine concentration was 34 µmol/L.\nThe platelet count fell to 138 four weeks after the last eculizumab dose and remained in the range of 98–160 × 109/L for the next 2 months. The plasma creatinine remained stable for 6 weeks, and then rose to 44 µmol/L. Two months after discontinuing the eculizumab, the plasma LDH had risen to 807 iu/L. The serum haptoglobin concentration was mildly reduced at 0.4 g/L and a blood film showed scanty red cell fragments. The plasma concentration of the terminal complement complex (sC5b-9) was elevated at 137 ng/mL (normal range <80). A renal biopsy was therefore performed.\nOf the 20 glomeruli in the light microscopy sections, five were globally sclerosed, and two showed periglomerular fibrosis. The patent glomeruli showed an increase in mesangial cells and matrix with segmental sclerosis and capsular adhesions. There were no platelet thrombi and extraglomerular vessels appeared normal. Immunostains for IgA, IgG, IgM and C3 were negative. There was focal tubular atrophy and interstitial fibrosis around damaged glomeruli. The toludine blue preparation and electron microscopy (EM) showed thickened glomerular capillary loops (double contour appearance), and areas of widening of the sub-endothelial space were seen. This space was filled with electron-lucent ‘fluffy’ material. This marked widening of the sub-endothelial space and fibrillar/particulate material is characteristic of HUS ().\nThe haematological, biochemical and histopathological features suggested active HUS, and eculizumab was therefore recommenced with an initial dose of 600 mg given 105 days after the last dose. Before restarting the eculizumab, the GFR was measured at 54 mL/min/1.73 m2, the serum complement C3 concentration was 0.9 g/L and the C4 was 0.25 g/L. The total haemolytic complement (CH50) was 111%, but the alternative pathway haemolytic complement (AP50) was low at 34% (normal 80–200). The haptoglobin was low at 0.4 g/L and rose to 0.96 g/L a week after the first dose. After 7 months of eculizumab therapy, the patient had normal haemoglobin (122 g/L), normal platelets (248 × 109/L) and a plasma creatinine concentration of 32 µmol/L. The GFR had risen to 71 mL/min/1.73 m2. The plasma concentration of sC5b-9 remained elevated at 133 ng/mL, casting doubt on the suitability of this assay for assessing activity and response to treatment.\nDNA extracted from a buccal smear at the time of diagnosis of HUS was amplified by PCR for all coding exons ± 10 bases of the CFH (factor H), CFI (factor I), C3 (complement component 3), CFB (factor B) and CD46 (membrane cofactor protein, MCP) genes. Amplified products were subjected to bi-directional Sanger sequencing (ABI), as previously described []. CD46 expression on cell surfaces was assessed on peripheral blood neutrophils after recovery from myelosuppression by flow cytometry using CD46 fluorescein isothiocyanate-conjugated antibody (BD Pharmingen, 555949).\nAn intronic sequence variant (c.1027+5G>T) was identified in CD46. This variant had not been previously reported nor was it detected in DNA from 188 normal control subjects. In silico splicing prediction analysis undertaken using Alamut (Interactive Bioscience Software, Seine Biopolis, 70 route de Lyons-la-Foret, 76 000 Rouen, France; ) suggests that this variant may cause aberrant splicing. Flow cytometry analysis showed reduced CD46 expression on neutrophils compatible with haploinsufficiency. After discussion with the parents, a decision was made not to test other family members, because the result was felt to be of limited clinical utility, outweighed by potential anxiety induced in a carrier.